Crystalline anti-htnfalpha antibodies

ABSTRACT

The present invention relates to a batch crystallization method for crystallizing an anti-hTNFalpha antibody which allows the production of said antibody on an industrial scale; antibody crystals as obtained according to said method; compositions containing said crystals as well as methods of use of said crystals and compositions.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/225,281, filed Sep. 2, 2011, which is a continuation applicationof U.S. application Ser. No. 11/977,677, filed Oct. 25, 2007, now U.S.Pat. No. 8,034,906, issued Oct. 11, 2011, which in turn claims priorityto U.S. Provisional Application Ser. No. 60/855,104, filed Oct. 27,2006. The entire contents of each of these applications are expresslyincorporated herein by reference.

The present invention relates to a batch crystallization method forcrystallizing an anti-hTNFalpha antibody which allows the production ofsaid antibody on an industrial scale; antibody crystals as obtainedaccording to said method; compositions containing said crystals as wellas methods of use of said crystals and compositions.

TECHNICAL BACKGROUND a) Antibody Crystals

With over 100 monoclonal antibodies currently being evaluated inclinical study phases 2 or 3, the mAb market can be considered one ofthe most promising biopharmaceutical markets. As these drugs have to bedelivered in single doses often exceeding 100 mg, there is an urgentneed to find suitable formulation strategies satisfying stability,safety and patient compliance.

However, highly concentrated liquid mAb formulations show increasedviscosity, hindering syringe ability through patient friendly thinneedles. Furthermore, the aggregation tendency of mAb molecules at suchhigh concentrations exponentially increases when compared to moderatelyconcentrated solutions. This is unacceptable, in all means, regardingsafety and stability requirements.

Thus, the delivery of high mAb doses is restrained to large volumes,which generally have to be delivered via infusion. This way of dosing iscost intensive and significantly reduces the patient's compliance.

Therefore, pharmaceutically applicable low volume mAb crystalsuspensions for subcutaneous injection would be highly desirable.Theoretically, degradation pathways influencing the mAb integrity shouldbe significantly decelerated due to the rigidity of a crystal lattice,where motions in the protein structure are hindered. Moreover, it can beexpected that the increase in viscosity would be significantly reducedwhen comparing highly concentrated crystal suspensions with liquidformulations. With respect to sustained release, it might be possible togenerate or alter protein crystals in that way that they dissolve slowlywhen brought into the patient's body. This would be a very elegant wayto deliver a sustained release formulation, as the extensive use ofexcipients and processes harming the mAb structure would be prevented.

Despite the great potential in using protein crystals as drug substance,few attempts have been made to systematically evaluate this strategy.

A well-known exemption is insulin, which was successfully crystallizeddecades ago. Today, the use of crystal suspensions of insulin is welldescribed, offering stable and long acting formulations being wellestablished on the market. The discrepancy between the development ofinsulin crystals and crystallization of all other proteins might berelated to the fact that ordered insulin aggregates are natively formedin the pancreas. Thus, insulin crystals are easily obtained when insulinis brought in contact with an excess of zinc ions. Most other proteinstend to form unordered precipitates rather than crystals, and therefore,finding crystallization conditions for a protein is a time consuming,non-trivial task.

Despite a great interest in harvesting protein crystals for x-raydiffraction analysis, the quest of finding suitable crystallizationconditions still is an empirical science, as in principle any proteinbehaves differently. To date, no general rule has been found which mightreliably predict by reason alone a successful crystallization conditionfor a protein of choice. Thus, obtaining crystals of a given proteinalways is referred to be the “bottle neck” of whatever intendedapplication is planned later on.

To make things even more challenging, antibodies are described to beespecially hard to crystallize, due to the flexibility of the molecule.

Nevertheless, examples of immunoglobulin crystals have been known for along time. The first example of immunoglobulin crystals were described150 years ago by an English physician, Henry Bence Jones; the isolatedcrystals of an abnormal Ig light chain dimer from the urine of a myelomapatient (Jones 1848). Such abnormal Igs have been known ever since asBence Jones proteins. In 1938, the spontaneous crystallization of adistinct abnormal Ig from the serum of a myeloma patient was described(von Bonsdorf, Groth et al. 1938), apparently an Ig heavy chain oligomer(MW 200 kDa).

Crystalline human immunoglobulins of normal structure (two heavy chainslinked to two light chains) were described over the next thirty years,again mostly isolated from myeloma patients (Putnam 1955). Davies andco-workers were the first to characterize the structure of an intacthuman myeloma antibody, named “Dob”, using x-ray crystallography (Terry,Matthews et al. 1968), and they determined its three-dimensionalstructure in 1971 (Sarma, Silverton et al. 1971). Their pioneering workwas followed by that of others, yielding the crystal structures of theIgG “Kol” (Huber, Deisenhofer et al. 1976), the IgG “Mcg” (Rajan, Ely etal. 1983), and a canine lymphoma IgG2a (Harris, Larson et al. 1992).

Crystals of immunoglobulins retain their distinctive immunologicalactivities upon re-dissolution. Nisonoff et al. reported in 1968 on arabbit anti-p-azobenzoate antibody, “X4”, that was easily crystallized.Antibody X4 was extensively characterized before crystallization as wellas after re-dissolution of the crystals. [¹²⁵I]-p-iodobenzoate was foundto bind specifically and potently to re-dissolved X4; the re-dissolvedcrystals also exhibited multiple specific Ouchterlony immunodiffusionreactions typical of the unpurified rabbit serum (Nisonoff, Zappacostaet al. 1968). Connell and co-workers described a human myelomagamma-immunoglobulin-1 kappa (IgG-κ), called “Tem”, that crystallizedspontaneously from serum at cold temperatures (Connell, Freedman et al.1973). Tem crystals were found to be well-formed and possessedrhombohedral symmetry. Tem-containing serum was extensivelycharacterized by agarose immunodiffusion techniques. Electrophoresis andimmunodiffusion of a re-dissolved solution of the Tem crystals showedthem to be identical with the material obtained from the serum bycryoprecipitation, and with the isolated myeloma protein (Connell,Freedman et al. 1973).

Mills and co-workers reported in 1983 an unusualcrystallocryoglobulinemia resulting from human monoclonal antibodies toalbumin (Mills, Brettman et al. 1983). Here, very similar cuboidalcrystals were isolated from two patients. Redissolution of the crystalsfollowed by electrophoresis and immunoelectrophoresis indicated that thecrystals were composed of two protein components, a monoclonalIgG-lambda and human serum albumin in a 1:2 ratio (Jentoft, Dearborn etal. 1982). The components were separated on preparative scale bydissolution of the original crystals followed by column chromatography.Although neither separated component crystallized on its own, uponrecombination the original bipartite complex reformed and thencrystallized. Further study of the distinctive sedimentationcharacteristics and immunological reactivity of the redissolved,separated IgG and its Fab fragment with human serum albumin indicatedthat reassociation of the two redissolved, separated components wasimmunologic in nature, i.e. that the crystalline antibody onceredissolved still possessed its native, highly specific (for human serumalbumin) binding characteristics (Mills, Brettman et al. 1983).

Recently. Margolin and co-workers reported on the potential therapeuticuses of crystalline antibodies (Yang, Shenoy et al. 2003). They foundthat the therapeutic monoclonal antibody trastuzumab (Herceptin®) couldbe crystallized (Shenoy, Govardhan et al. 2002). Crystalline trastuzumabsuspensions were therapeutically efficacious in a mouse tumor model,thus demonstrating retention of biological activity by crystallinetrastuzumab (Yang. Shenoy et al. 2003).

b) Crystallization Techniques

Unlike some other scientific or engineering endeavors, thecrystallization of diverse proteins cannot be carried out successfullyusing defined methods or algorithms. Certainly, there have been greattechnical advances in the last 20-30 years, as noted by theworld-renowned expert in protein crystallization, A. McPherson.McPherson provides extensive details on tactics, strategies, reagents,and devices for the crystallization of macromolecules. He does not,however, provide a method to ensure that any given macromolecule canindeed be crystallized by a skilled person with reasonable expectationof success. McPherson states for example: “Whatever the procedure, noeffort must be spared in refining and optimizing the parameters of thesystem, both solvent and solute, to encourage and promote specificbonding interactions between molecules and to stabilize them once theyhave formed. This latter aspect of the problem generally depends on thespecific chemical and physical properties of the particular protein ornucleic acid being crystallized.” (McPherson 1999, p. 159)

It is widely accepted by those skilled in the art of proteincrystallization that no algorithm exists to take a new protein ofinterest, apply definite process steps, and thereby obtain the desiredcrystals.

Several screening systems a commercially available (for example Hampton1 and 2, Wizzard I and II) which allow, on a microliter scale, to screenfor potentially suitable crystallization conditions for a specificprotein. However, positive results obtained in such a screening systemdo not necessarily allow successful crystallization in a larger,industrially applicable batch scale. Conversion of microliter-sizecrystallization trials into industrial dimensions is described to be achallenging task (see Jen et al., 2001).

Baldock et al (1996) reported on a comparison of microbatch and vapordiffusion for initial screening of crystallization conditions. Sixcommercially available proteins were screened using a set ofcrystallization solutions. The screens were performed using the mostcommon vapor diffusion method and three variants of a microbatchcrystallization method, including a novel evaporation technique. Out of58 crystallization conditions identified, 43 (74%) were identified bymicrobatch, while 41 (71%) were identified by vapor diffusion.Twenty-six conditions were found by both methods, and 17 (29%) wouldhave been missed if microbatch had not been used at all. This shows thatthe vapor diffusion technique, which is most commonly used in initialcrystallization screens does not guarantee positive results.

c) hTNFalpha Antibody Crystals

Human TNFalpha (hTNFalpha) is considered as a causative agent ofnumerous diseases. There is, therefore, a great need for suitablemethods of treating such hTNFalpha related disorders. One promisingtherapeutic approach comprises the administration of pharmaceuticallyeffective doses of anti-human TNFalpha antibodies. Recently one suchantibody, designated D2E7, or generically adalimumab, has been put onthe market and is commercialised under the trade name HUMIRA®.

WO-A-02/072636 disclosed the crystallization of the whole, intactantibodies Rituximab, Infliximab and Trastuzumab. Most of thecrystallization experiments were performed with chemicals with uncleartoxicity, like imidazole, 2-cyclohexyl-ethanesulfonate (CHES),methylpentanediol, copper sulphate, and 2-morpholino-ethanesulfonate(MES). Most of the examples used seed crystals to initiatecrystallization.

WO-A-2004/009776 disclosed crystallization experiments in the microliterscale using the sitting drop vapor diffusion technique by mixing equalvolumes (1 μl) of different crystallization buffers and D2E7 F(ab)′₂ orFab fragments. While several experimental conditions were reported foreach of said fragments, no successful crystallization of the whole,intact D2E7 antibody was reported.

Methods for preparing crystals of any given anti-human TNFalpha wholeantibodies, in particular of D2E7, therefore are not available.

The problem to be solved according to the present invention is,therefore, to develop suitable batch crystallization conditions foranti-hTNFalpha antibodies, in particular for the human anti-hTNFalphaantibody D2E7, and to establish crystallization process conditionsapplicable to volumes relevant for industrial antibody crystalproduction. At the same time a crystallization process should beestablished that does not make use of toxic agents, which mightnegatively affect the pharmaceutical applicability of such antibodies.

SUMMARY OF THE INVENTION

The above-mentioned problem was, surprisingly, solved by the findingthat it is possible to obtain crystals of a whole anti-hTNFalphaantibody in batch crystallization volumes above the microliter scale byapplying physiologically acceptable inorganic phosphate salts as thecrystallization-inducing agent.

PREFERRED EMBODIMENTS

In a first aspect the invention relates to a batch crystallizationmethod for crystallizing an anti-hTNFalpha antibody, comprising thesteps of:

-   a) providing an aqueous solution of said antibody in admixture with    an inorganic phosphate salt as crystallization agent, as for example    by mixing an aqueous solution of said antibody, wherein the antibody    preferably is present in dissolved form, with an aqueous    crystallization solution comprising an inorganic phosphate salt as    crystallization agent in dissolved form, or alternatively by adding    said crystallization agent in solid form; and-   b) incubating said aqueous crystallization mixture until crystals of    said antibody are formed.

The crystallization method of the invention generally is performed at apH of said aqueous crystallization mixture in the range of about pH 3 toabout 5, in particular about 3.5 to about 4.5, or about 3.7 to about4.2.

Moreover, said aqueous crystallization mixture may contain at least onebuffer. Said buffer may, in particular, comprise an acetate component asmain component, especially an alkali metal salt, in particular, sodiumacetate. Said salt is adjusted by addition of an acid, in particularacetic acid, to the required pH. In a preferred embodiment of thecrystallization method, the buffer concentration (total acetate) in saidaqueous crystallization mixture is 0 to about 0.5 M, or about 0.02 toabout 0.5 M, as for example about 0.05 to about 0.3 M, or about 0.15 toabout 0.2 M.

In a further particular embodiment of the crystallization methodaccording to the invention, the phosphate salt used as the precipitatingagent is selected from hydrogenphosphate salts, such as mono- ordihydrogenphosphate salts, in particular an ammonium salt or an alkalimetal salt, for example a salt containing Na⁺ or K⁺ ions, or a mixturethereof comprising of at least two different salts. Suitable examplesare: NaH₂PO₄, Na₂HPO₄, KH₂PO₄, K₂HPO₄, NH₄H₂PO₄, (NH₄)₂HPO₄, andmixtures thereof.

In particular, the phosphate salt concentration in the crystallizationmixture is in the range of about 1 to about 6 M, for example a range ofabout 1.0 to about 4.0 M, or about 1.0 to about 3.0 M, or about 1.5 toabout 2.8 M, or about 2.0 to about 2.6M.

In a preferred embodiment of the invention, protein solution andcrystallization solution are combined in a ratio of about 1:1. Thus,molarities of the buffering agents/crystallization agents in theoriginal crystallization solution are about double as high as in thecrystallization mixture.

Typically, the crystallization method is performed in a batch volume inthe range of about 1 ml to about 20000 l, or 1 ml to about 15000 l, or 1ml to about 12000 l, or about 1 ml to about 10000 l, or 1 ml to about6000 l, or 1 ml to about 3000 l, or 1 ml to about 1000 l, or 1 ml toabout 100 l, as for example about 50 ml to about 8000 ml, or about 100ml to about 5000 ml, or about 1000 ml to about 3000 ml; or about 1 l toabout 1000 l; or about 10 l to about 500 l.

In addition, the crystallization method of the invention may beperformed so that at least one of the following additionalcrystallization conditions is achieved:

-   a) incubation is performed for between about 1 hour to about 60    days, for example about 1 to about 30 days, or about 2 to 10 days;-   b) incubation is performed at a temperature between about 0° C. and    about 50° C., for example about 4° C. and about 37° C.;-   c) the antibody concentration (i.e. protein concentration) in the    crystallization mixture is in the range of about 1 to 200 mg/ml or 1    to 100 mg/ml, for example 1.5 to 20 mg/ml, in particular in the    range of about 2 to 15 mg/ml, or 5 to 10 mg/ml. The protein    concentration may be determined according to standard procedures for    protein determination.

According to a particularly preferred method, crystallization isperformed under the following conditions of the crystallization mixture:

Phosphate salt: NaH₂PO₄, 1.5 to 2.5 Mbuffer: total acetate, 0 to 0.3 MpH: 3.6 to 4.2anti-hTNFalpha concentration: 3 to 10 mg/ml

Temperature: 18 to 24° C.

Batch volume: 1 to 100 l

Agitation: None

Duration: 4 to 15 days

The crystallization mixtures as outlined above are usually obtained byadding a crystallization agent in solution or as solid to the proteinsolution. Both solutions may be, but do not have to be buffered.Crystallization agent molarity and buffer molarity in the originalcrystallization solution is usually higher than in the crystallizationmixture as it is “diluted” with the protein solution.

In a further embodiment, the crystallization method of the invention mayfurther comprise the step of drying the obtained crystals. Suitabledrying methods comprise evaporative drying, spray drying,lyophilization, vacuum drying, fluid bed drying, spray freeze drying,near critical drying, supercritical drying, and nitrogen gas drying.

In a further embodiment, the crystallization method of the invention mayfurther comprise the step of exchanging the crystallization motherliquor with a different buffer, e.g. a buffer containing polyethyleneglycol (PEG) with a molar mass in the range of about 300 to 8000 Daltonsor mixtures of PEGs, by centrifugation, diafiltration, ultrafiltrationor other commonly used buffer exchange techniques.

The present invention also relates to a crystal of an anti-hTNFalphaantibody, obtainable by a crystallization method as defined above and ingeneral to crystals of an anti-hTNFalpha antibody

The crystals of the invention are typically characterized by aneedle-like morphology with a maximum length l of about 2-500 μm orabout 100-300 μm and an l/d ratio of about 3 to 30, but may also haveother geometrical appearances

Said crystal may be obtained from a polyclonal antibody or, preferably,a monoclonal antibody.

In particular, said antibody is selected from the group consisting of:non-chimeric or chimeric antibodies, humanized antibodies,non-glycosylated antibodies, human antibodies and mouse antibodies. Inparticular the antibody to be crystallized is a non-chimeric, humanantibody optionally further processed for improving the antigen-binding.

Preferably, said crystals are obtained from an IgG antibody such as, forexample, an IgG1, IgG2, IgG3 or IgG4 antibody. In particular, saidantibody is a whole anti-human TNFalpha antibody of the group IgG1.

In a preferred embodiment, the crystals are prepared from an isolatedhuman antibody, that dissociates from hTNFalpha with a Kd of 1×10⁻⁸ M orless, more preferably 1×10⁻⁹ M or less, and even more preferably 5×10⁻¹⁰M or less, and a K_(a) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance, and neutralizes hTNFalphacytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ Mor less.

In particular, said crystals may be prepared from an isolated humanantibody with the following characteristics: a) dissociates from humanTNFalpha with a k_(off) rate constant of 1×10⁻³ s⁻¹ or less, asdetermined by surface plasmon resonance; b) has a light chain CDR3domain comprising the amino acid sequence of SEQ ID NO: 3, or modifiedfrom SEQ ID NO: 3 by a single alanine substitution at position 1, 4, 5,7 or 8, or by one to five conservative amino acid substitutions atpositions 1, 3, 4, 6, 7, 8 and/or 9; c) has a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 4, or modified from SEQID NO: 4 by a single alanine substitution at position 2, 3, 4, 5, 6, 8,9, 10 or 11, or by one to five conservative amino acid substitutions atpositions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFalpha with a k_(off) of 5×10⁻⁴ s⁻¹ or less.Even more preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFalpha with a k_(off) of 1×10⁻⁴ s⁻¹ or less.

In a particularly preferred embodiment, said crystals are prepared froman isolated human antibody with a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chainvariable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2.

Most preferred are crystals prepared from the antibody D2E7, asdisclosed in WO-A-97/29131 or a functional equivalent thereof. Saidantibody is recombinantly produced in Chinese hamster ovary cells andcomprises a heavy chain sequence according to SEQ ID NO:6 and a lightchain sequence according to SEQ ID NO: 5.

In a further embodiment, the invention relates to a solid, liquid orsemi-solid pharmaceutical composition comprising: (a) crystals of ananti-hTNFalpha antibody as defined in any one of claims 15 to 26, and(b) at least one pharmaceutically acceptable excipient stablymaintaining the antibody crystals.

Another aspect of this invention relates to a solid, liquid orsemi-solid pharmaceutical composition comprising: (a) crystals of ananti-hTNFalpha antibody as defined herein, and (b) at least onepharmaceutically acceptable excipient encapsulating or embedding saidantibody crystals. The composition may further comprise (c) at least onepharmaceutically acceptable excipient stably maintaining the antibodycrystals. Moreover, encapsulation and embedding may be implemented inconjunction.

In particular, said compositions may have an antibody crystalconcentration higher than about 1 mg/ml, in particular about 200 mg/mlor more, for example about 200 to about 600 mg/ml, or about 300 to about500 mg/ml.

Said excipients may comprise at least one polymeric, optionallybiodegradable carrier or at least one oil or lipid carrier.

Said polymeric carrier may be a polymer selected from one or more of thegroup consisting of: poly(acrylic acid), poly(cyanoacrylates),poly(amino acids), poly(anhydrides), poly(depsipeptide), poly(esters),poly(lactic acid), poly(lactic-co-glycolic acid) or PLGA,poly(β-hydroxybutryate), poly(caprolactone), poly(dioxanone);poly(ethylene glycol), poly(hydroxypropyl) methacrylamide,poly(organo)phosphazene, poly(ortho esters), poly(vinyl alcohol),poly(vinylpyrrolidone), maleic anhydride alkyl vinyl ether copolymers,pluronic polyols, albumin, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends andcopolymers thereof.

Said oil (or oily liquid) may be an oil (or oily liquid) selected fromone or more of the group consisting of: oleaginous almond oil, corn oil,cottonseed oil, ethyl oleate, isopropyl myristate, isopropyl palmitate,mineral oil, light mineral oil, octyldodecanol, olive oil, peanut oil,persic oil, sesame oil, soybean oil, squalane, liquid triglycerides,liquid waxes, higher alcohols.

Said lipid carrier may be a lipid selected from one or more of the groupconsisting of: fatty acids and salts of fatty acids, fatty alcohols,fatty amines, mono-, di-, and triglycerides of fatty acids,phospholipids, glycolipids, sterols and waxes and related similarsubstances. Waxes are further classified in natural and syntheticproducts. Natural materials include waxes obtained from vegetable,animal or minerals sources such as beeswax, carnauba or montanwax.Chlorinated naphthalenes and ethylenic polymers are examples forsynthetic wax products.

In a preferred embodiment, said composition is an injectable compositioncomprising anti-hTNFalpha antibody crystals as defined above and havingan antibody crystal concentration in the range of about 10 to about 400or about 50 to about 300 mg/ml.

In a further aspect the invention relates to a crystal slurry comprisinganti-hTNFalpha antibody crystals as defined above having an antibodycrystal concentration higher than about 100 mg/ml, for example about 150to about 600 mg/ml, or about 200 to about 400 mg/ml.

The present invention also relates to a method for treating a mammalcomprising the step of administering to the mammal an effective amountof whole anti-hTNFalpha antibody crystals as defined above or aneffective amount of the composition as defined above. Preferably, saidcomposition is administered by parenteral route, oral route, or byinjection.

Furthermore, the present invention relates to a method of treating ahTNFalpha-related disorder in a subject that comprises administering atherapeutically effective amount of antibody crystals as defined above.

In particular, said hTNFalpha-related disorder is selected from:

an autoimmune disease, in particular rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis and gouty arthritis, an allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis and nephroticsyndrome; an infectious disease, transplant rejection orgraft-versus-host disease, malignancy, pulmonary disorder, intestinaldisorder, cardiac disorder, inflammatory bone disorders, bone resorptiondisease, alcoholic hepatitis, viral hepatitis, fulminant hepatitis,coagulation disturbances, burns, reperfusion injury, keloid formation,scar tissue formation, pyrexia, periodontal disease, obesity andradiation toxicity; a spondyloarthropathy, a pulmonary disorder, acoronary disorder, a metabolic disorder, anemia, pain, a hepaticdisorder, a skin disorder, a nail disorder, or vasculitis, Behcet'sdisease, ankylosing spondylitis, asthma, chronic obstructive pulmonarydisease (COPD), idiopathic pulmonary fibrosis (IPF), restenosis,diabetes, anemia, pain, a Crohn's disease-related disorder, juvenilerheumatoid arthritis (JRA), a hepatitis C virus infection, psoriasis,psoriatic arthritis, chronic plaque psoriasis, age-related cachexia,Alzheimer's disease, brain edema, inflammatory brain injury, chronicfatigue syndrome, dermatomyositis, drug reactions, edema in and/oraround the spinal cord, familial periodic fevers, Felty's syndrome,fibrosis, glomerulonephritides (e.g. post-streptococcalglomerulonephritis or IgA nephropathy), loosening of prostheses,microscopic polyangiitis, mixed connective tissue disorder, multiplemyeloma, cancer and cachexia, multiple organ disorder, myelo dysplasticsyndrome, orchitism osteolysis, pancreatitis, including acute, chronic,and pancreatic abscess, periodontal disease polymyositis, progressiverenal failure, pseudogout, pyoderma gangrenosum, relapsingpolychondritis, rheumatic heart disease, sarcoidosis, sclerosingcholangitis, stroke, thoracoabdominal aortic aneurysm repair (TAAA), TNFreceptor associated periodic syndrome (TRAPS), symptoms related toYellow Fever vaccination, inflammatory diseases associated with the ear,chronic ear inflammation, or pediatric ear inflammation, uveitis,sciatica, prostatitis, endometriosis, choroidal neovascularization,lupus, Sjogren's syndrome, and wet macular degeneration.

Moreover, the present invention relates to the use of wholeanti-hTNFalpha antibody crystals as defined above for preparing apharmaceutical composition for treating a hTNFalpha-related disease asdefined above.

Finally, the present invention provides anti-hTNFalpha antibody crystalsas defined above for use in medicine.

DESCRIPTION OF FIGURES

FIG. 1: D2E7 crystals from Example 37 after 6 days.

FIG. 2: D2E7 crystals manufactured in 1 mL batch volume, ambienttemperature.

FIG. 3: D2E7 crystals manufactured in 50 mL batch volume, ambienttemperature.

FIG. 4: D2E7 crystals manufactured in 10 L batch volume, ambienttemperature.

FIG. 5: D2E7 crystals manufactured according to the invention andbirefringence thereof. FIG. 5A shows clusters of Adalimumab needle-likecrystals and was photographed under crossed polarizers. FIG. 5B is takenwith plane polars and shows the particle morphology. FIG. 5C showsbirefringence and was taken with crossed polar and a red compensator orquarter wave plate. FIG. 5D shows birefringence and was taken withcrossed polars.

FIG. 6: D2E7 crystal suspensions at different concentrations injectedvia different gauge needles.

FIG. 7: FT-IR analysis of D2E7 crystal suspension.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

A “batch method of crystallization” comprises the step of adding thecrystallization solution comprising the crystallization agent,preferably in dissolved form, to the solution of the antibody to becrystallized.

A “micro scale crystallization method”, which may for example be basedupon vapor diffusion, comprises the steps of admixing a small volume ofantibody solution in the microliter range with a reservoir buffercontaining a crystallization agent; placing a droplet of said mixture ina sealed container adjacent to an aliquot of said reservoir buffer;allowing exchange of solvent between the droplet and the reservoir byvapor diffusion, during which the solvent content in said dropletchanges and crystallization may be observed if suitable crystallizationconditions are reached.

A “crystallization agent”, in the present case a phosphate salt, favorscrystal formation of the antibody to be crystallized.

A “crystallization solution” contains said crystallization agent indissolved form. Preferably said solution is an aqueous system, i.e. theliquid constituents thereof predominantly, consist of water. As forexample, 80 to 100 wt.-% or 95 to 100 wt.-% or 98 to 100 wt.-% may bewater.

Antibody “crystals” are one form of the solid state of matter of saidprotein, which is distinct from a second solid form, i.e. the amorphousstate, which exists essentially as an unorganized, heterogeneous solid.Crystals have a regular three-dimensional structure, typically referredto as a lattice. An antibody crystal comprises a regularthree-dimensional array of antibody molecules. See Giege, R. andDucruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, aPractical Approach, 2nd ed., pp. 1-16, Oxford University Press, New York(1999).

A “whole” or “intact” anti-hTNFalpha antibody as crystallized accordingto this invention, is a functional antibody that is able to recognizeand bind to its antigen human TNFalpha in vitro and/or in vivo. Theantibody may initiate subsequent immune system reactions of a patientassociated with antibody-binding to its antigen, in particular DirectCytotoxicity, Complement-Dependent Cytotoxicity (CDC), andAntibody-Dependent Cytotoxicity (ADCC). The antibody molecule has astructure composed of two identical heavy chains (MW each about 50 kDa)covalently bound to each other, and two identical light chains (MW eachabout 25 kDa), each covalently bound to one of the heavy chains. Thefour chains are arranged in a classic “Y” motif. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The complete antibody molecule has two antigen binding sites,i.e. is “bivalent”. The two antigen binding sites are specific for onehTNFalpha antigen, i.e. the antibody is “mono-specific”.

“Monoclonal antibodies” are antibodies that are derived from a singleclone of B lymphocytes (B cells), and recognize the same antigenicdeterminant. Whole monoclonal antibodies are those that have theabove-mentioned classic molecular structure that includes two completeheavy chains and two complete light chains. Monoclonal antibodies areroutinely produced by fusing the antibody-producing B cell with animmortal myeloma cell to generate B cell hybridomas, which continuallyproduce monoclonal antibodies in cell culture. Other production methodsare available, as for example expression of monoclonal antibodies inbacterial, yeast, insect, or mammalian cell culture using phage-displaytechnology; in vivo production in genetically modified animals, such ascows, goats, pigs, rabbits, chickens, or in transgenic mice which havebeen modified to contain and express the entire human B cell genome; orproduction in genetically modified plants, such as tobacco and corn.Anti-hTNFalpha antibodies from all such sources may be crystallizedaccording to this invention.

The monoclonal antibodies to be crystallized according to the inventioninclude “chimeric” anti-hTNFalpha antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass. As an example there maybe mentioned a mouse/human chimera containing variable antigen-bindingportions of a murine antibody and constant portions derived from a humanantibody.

“Humanized” forms of non-human (e.g. murine) anti-hTNFalpha antibodiesare also encompassed. These are chimeric antibodies that contain minimalsequence derived from a non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins in which residues from acomplementarity determining region (CDR) or hypervariable loop (HVL) ofthe human immunoglobulin are replaced by residues from a CDR or HVL of anon-human species, such as mouse, rat, rabbit or non-human primate,having the desired functionality. Framework region (FR) residues of thehuman immunoglobulin may replaced by corresponding non-human residues toimprove antigen binding affinity. Furthermore, humanized antibodies maycomprise residues that are found neither in the corresponding human ornon-human antibody portions. These modifications may be necessary tofurther improve antibody efficacy.

A “human antibody” or “fully human antibody” is one, which has an aminoacid sequence which corresponds to that of an antibody produced by ahuman or which is recombinantly produced. The term “human antibody”, asused herein, is intended to include antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g. mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library,antibodies isolated from an animal (e.g. a mouse) that is transgenic forhuman immunoglobulin genes (see e.g. Taylor, L. D., et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFalpha □activity”), is intended to refer to an antibodywhose binding to hTNFalpha results in inhibition of the biologicalactivity of hTNFalpha. This inhibition of the biological activity ofhTNFalpha can be assessed by measuring one or more indicators ofhTNFalpha biological activity, such as hTNFalpha-induced cytotoxicity(either in vitro or in vivo), hTNFalpha-induced cellular activation andhTNFalpha binding to hTNFalpha receptors. These indicators of hTNFalphabiological activity can be assessed by one or more of several standardin vitro or in vivo assays known in the art. Preferably, the ability ofan antibody to neutralize hTNFalpha activity is assessed by inhibitionof hTNFalpha-induced cytotoxicity of L929 cells. As an additional oralternative parameter of hTNFalpha activity, the ability of an antibodyto inhibit hTNFalpha-induced expression of ELAM-1 on HUVEC, as a measureof hTNFalpha-induced cellular activation, can be assessed.

An “affinity matured” anti-hTNFalpha antibody is one with one or morealterations in one or more hypervariable regions, which result in animprovement in the affinity of the antibody for antigen, compared to aparent antibody. Affinity matured antibodies will have nanomolar or evenpicomolar affinities values for the target antigen. Affinity maturedantibodies are produced by procedures known in the art. Marks et al.,Bio/Technology 10:779-783 (1992) describes affinity maturation by VH andVL domain shuffling. Random mutagenesis of CDR and/or framework residuesis described by: Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813(1994); Scier et al., Gene 169:147-155 (1995); Yelton et al., J.Immunol. 155:1994-2004 (1995): Jackson et al., J. Immunol. 154(7):3310-9(1995); and Hawkins et al., J. Mol Biol. 226:889-896 (1992).

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g. an isolated antibody that specificallybinds hTNFalpha is substantially free of antibodies that specificallybind antigens other than hTNFalpha). An isolated antibody thatspecifically binds hTNFalpha may, however, have cross-reactivity toother antigens, such as hTNFalpha molecules from other species.Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The term “human TNFalpha” (abbreviated herein as hTNFalpha, or simplyhTNF), as used herein, is intended to refer to a human cytokine thatexists as a 17 kDa secreted form and a 26 kDa membrane-associated form,the biologically active form of which is composed of a trimer ofnoncovalently bound molecules. The structure of hTNFalpha is describedfurther in, for example, Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFalpha is intended toinclude recombinant human TNFalpha (rhTNFalpha), which can be preparedby standard recombinant expression methods or purchased commercially (R& D Systems, Catalog No. 210-TA, Minneapolis, Minn.).

The term “k_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

A “functional equivalent” of a specific “parent” anti-hTNFalpha antibodyas crystallized according to the invention is one which shows the sameantigen-specificity, differs however with respect to the molecularcomposition of the “parent” antibody on the amino acid level orglycosylation level. Said differences, however, may be merely such thatthe crystallization conditions do not deviate from the parameter rangesas disclosed herein.

“Encapsulation” of antibody crystals refers to a formulation where theincorporated crystals are individually coated by at least one layer of acoating material. In a preferred embodiment, such coated crystals mayhave a sustained dissolution rate.

“Embedding” of antibody crystals refers to a formulation where thecrystals, which might be encapsulated or not, are incorporated into asolid, liquid or semi-solid carrier in a disperse manner. Such embeddedcrystallized antibody molecules may be released or dissolved in acontrolled, sustained manner from the carrier.

B. Method of Crystallization

The crystallization method of the invention is in principle applicableto any anti-hTNFalpha antibody. Said antibody may be a polyclonalantibody or, preferably, a monoclonal antibody. Said antibody may bechimeric antibodies, humanized antibodies, human antibodies ornon-human, as for example mouse antibodies, each in glycosylated ornon-glycosylated form. In particular the method is applicable to D2E7and functional equivalents thereof.

Preferably said anti-hTNFalpha antibody is an IgG antibody, inparticular an anti human TNFalpha antibody of the group IgG1.

Unless otherwise stated the crystallization method of the inventionmakes use of technical equipment, chemicals and methodologies well knownin the art. However, as explained above, the present invention is basedon the surprising finding that the selection of specific crystallizationconditions, in particular, the selection of specific crystallizationagents, optionally further combined with specific pH conditions and/orconcentration ranges of the corresponding agents (buffer, antibody,crystallization agent), allows for the first time to preparereproducibly and in a large scale stable crystals of antibodies, inparticular non-chimeric, human antibodies, directed against hTNF alpha,which can be further processed to form an active ingredient of asuperior, highly advantageous pharmaceutical composition.

The starting material for performing the crystallization method normallycomprises a concentrated solution of the antibody to be crystallized.The protein concentration may, for example, be in the range of about 5to 75 mg/ml. Said solution may contain additives stabilizing saiddissolved antibody, and it may be advisable to remove said additives inadvance. This can be achieved by performing a buffer exchange step.

Preferably said starting material for performing the crystallizationcontains the antibody in an aqueous solution, having a pH adjusted inthe range of about 3.2 to 8.2, or about 4.0 to 8.0, in particular about4.5 to 6.5, preferably around 5.0 to 5.5. The pH may be adjusted bymeans of a suitable buffer applied in a final concentration of about 1to 50 mM, in particular about 1 to 10 mM. The solution may containadditives, as for example in a proportion of about 0.01 to 15, or 0.1 to5, or 0.1 to 2 wt.-% based on the total weight of the solution, likesalts, sugars, sugar alcohols and surfactants, in order to furtherstabilize the solution. The excipients should preferably be selectedfrom physiologically acceptable compounds, routinely applied inpharmaceutical preparations. As non-limiting examples there may bementioned salts, like NaCl; surfactants, like polysorbate 80 (Tween 80),polysorbate 20 (Tween 20); sugars, like sucrose, trehalose; sugaralcohols, like mannitol, sorbitol; and buffer agents, likephosphate-based buffer systems, as sodium and potassium hydrogenphosphate buffers as defined above, acetate buffer, phosphate buffer,citrate buffer, TRIS buffer, maleate buffer or succinate buffer,histidine buffer; amino acids, like histidine, arginine and glycine.

The buffer exchange may be performed by means of routine methods, forexample dialysis or ultrafiltration.

The initial protein concentration of the aqueous solution used asstarting material should be in the range of about 0.5 to about 200 orabout 1 to about 50 mg/ml.

Depending on the intended final batch size (which may be in the range of1 ml to 20000 (twenty thousand) litres) an initial volume of saidaqueous antibody solution is placed in an appropriate container (as forexample a vessel, bottle or tank) made of inert material, as for exampleglass, polymer or metal. The initial volume of said aqueous solution maycorrespond to about 30 to 80%, normally about 50% of the final batchsize.

If necessary the solution after having been filled into said containerwill be brought to standardized conditions. In particular, thetemperature will be adjusted in the range of about 4° C. and about 37°C.

Then the crystallization solution, containing the crystallization agentin an appropriate concentration, optionally pre-conditioned in the sameway as the antibody solution, is added to the antibody solution.

The addition of the crystallization solution is performed continuouslyor discontinuously optionally under gentle agitation in order tofacilitate mixing of the two liquids. Preferably the addition isperformed under conditions where the protein solution is provided underagitation and the crystallization solution (or agents in its solid from)is/are added in a controlled manner.

The formation of the antibody crystals is initiated by applying aphosphate salt, in particular a hydrogen phosphate salt, and preferablyan alkali metal salt, or a mixture of at least two different alkalimetal salts as defined above as the crystallization agent. Thecrystallization solution contains the agent in a concentration which issufficient to afford a final concentration of the phosphate salt in saidcrystallization mixture in the range of about 1 to 6 M.

Preferably, the crystallization solution additionally contains an acidicbuffer, i.e. different from that of the antibody solution, in aconcentration suitable to allow the adjustment of the pH of thecrystallization mixture in the range of about 3 to 5.

After having finished the addition of said crystallization solution, thethus obtained mixture may be further incubated for about 1 hour to about60 days in order to obtain a maximum yield of antibody crystals. Ifappropriate, the mixture may, for example, be agitated, gently stirred,rolled or moved in a manner known per se.

Finally, the crystals obtained may be separated by known methods, forexample filtration or centrifugation, as for example by centrifugationat about 200-20000 rpm, preferably 500-2000 rpm, at room temperature or4° C. The remaining mother liquor may be discarded or further processed.

If necessary, the thus isolated crystals may be washed and subsequentlydried, or the mother liquor can be exchanged by a different solventsystem suitable for storage and/or final use of the antibodies suspendedtherein.

Antibody crystals formed according to the present invention may vary intheir shape. Shapes typically may include needles, cone-like, sphericaland sea urchin like shapes. The size of the crystals can be on the orderof higher nm to mm size (as for example length). In some embodiments,the crystals are at least about 10 μm in size, and may be visible to thenaked eye. For therapeutic administration, the size of the crystals willvary depending on the route of administration, for example, forsubcutaneous administration the size of the crystals may be larger thanfor intravenous administration.

The shape of the crystals may be altered by adding specific additionaladditives to the crystallization mixture, as has been previouslydescribed for both protein crystals and crystals of low molecular weightorganic and inorganic molecules.

If necessary, it may be verified that the crystals are in fact crystalsof said antibody. Crystals of an antibody can be analyzedmicroscopically for birefringence. In general, crystals, unless of cubicinternal symmetry, will rotate the plane of polarization of polarizedlight. In yet another method, crystals can be isolated, washed,resolubilized and analyzed by SDS-PAGE and, optionally, stained with ananti-Fc receptor antibody. Optionally, the resolubilized antibody canalso be tested for binding to its hTNFalpha utilizing standard assays.

Crystals as obtained according to the invention may also be crosslinkedto one another. Such crosslinking may enhance stability of the crystals.Methods for crosslinking crystals described, for example, in U.S. Pat.No. 5,849,296. Crystals can be crosslinked using a bifunctional reagentsuch as glutaraldehyde. Once crosslinked, crystals can be lyophilizedand stored for use, for example, in diagnostic or therapeuticapplications.

In some cases, it may be desirable to dry the crystal. Crystals may bedried by means of inert gases, like nitrogen gas, vacuum oven drying,lyophilization, evaporation, tray drying, fluid bed drying, spraydrying, vacuum drying or roller drying. Suitable methods are well known.

Crystals formed according to the invention can be maintained in theoriginal crystallization solution, or they can be washed and combinedwith other substances, like inert carriers or ingredients to formcompositions or formulations comprising crystals of the invention. Suchcompositions or formulations can be used, for example, in therapeuticand diagnostic applications.

A preferred embodiment is to combine a suitable carrier or ingredientwith crystals of the invention in that way that crystals of theformulation are embedded or encapsulated by an excipient. Suitablecarriers may be taken from the non limiting group of: poly(acrylicacid), poly(cyanoacrylates), poly(amino acids), poly(anhydrides),poly(depsipeptide), poly(esters), poly(lactic acid),poly(lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutryate),poly(caprolactone), poly(dioxanone); poly(ethylene glycol),poly(hydroxypropyl) methacrylamide, poly(organo)phosphazene, poly(orthoesters), poly(vinyl alcohol), poly(vinylpyrrolidone), maleic anhydridealkyl vinyl ether copolymers, pluronic polyols, albumin, alginate,cellulose and cellulose derivatives, collagen, fibrin, gelatin,hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof, SAIB, fatty acids andsalts of fatty acids, fatty alcohols, fatty amines, mono-, di-, andtriglycerides of fatty acids, phospholipids, glycolipids, sterols andwaxes and related similar substances. Waxes are further classified innatural and synthetic products. Natural materials include waxes obtainedfrom vegetable, animal or minerals sources such as beeswax, carnauba ormontanwax. Chlorinated naphthalenes and ethylenic polymers are examplesfor synthetic wax products.

C. Compositions

Another aspect of the invention relates to compositions/formulationscomprising anti-hTNFalpha antibody crystals in combination with at leastone carrier/excipient.

The formulations may be solid, semisolid or liquid.

Formulations of the invention are prepared, in a form suitable forstorage and/or for use, by mixing the antibody having the necessarydegree of purity with a physiologically acceptable additive, likecarrier, excipient and/or stabilizer (see for example Remington'sPharmaceutical Sciences, 16th Edn., Osol, A. Ed. (1980)), in the form ofsuspensions, lyophilized or dried in another way. Optionally furtheractive ingredients, as for example different antibodies, biomolecules,chemically or enzymatically synthesized low-molecular weight moleculesmay be incorporated as well.

Acceptable additives are non-toxic to recipients at the dosages andconcentrations employed. Nonlimiting examples thereof include:

-   -   Acidifying agents, like acetic acid, citric acid, fumaric acid,        hydrochloric acid, malic acid, nitric acid, phosphoric acid,        diluted phosphoric acid, sulfuric acid, tartaric acid.    -   Aerosol propellants, like butane, dichlorodifluoromethane,        dichlorotetrafluoroethane, isobutane, propane,        trichloromonofluormethane.    -   Air displacements, like carbon dioxide, nitrogen;    -   Alcohol denaturants, like methyl isobutyl ketone, sucrose        octacetate;    -   Alkalizing agents, like ammonia solution, ammonium carbonate,        diethanolamine, diisopropanolamine, potassium hydroxide, sodium        bicarbonate, sodium borate, sodium carbonate, sodium hydroxide,        trolamine;    -   Antifoaming agents, like dimethicone, simethicone.    -   Antimicrobial preservatives, like benzalkonium chloride,        benzalkonium chloride solution, benzethonium chloride, benzoic        acid, benzyl alcohol, butylparaben, cetylpyridinium chloride,        chlorobutanol, chlorocresol, cresol, dehydroacetic acid,        ethylparaben, methylparaben, methylparaben sodium, phenol,        phenylethyl alcohol, phenylmercuric acetate, phenylmercuric        nitrate, potassium benzoate, potassium sorbate, propylparaben,        propylparaben sodium, sodium benzoate, sodium dehydroacetate,        sodium propionate, sorbic acid, thimerosal, thymol.    -   Antioxidants, like ascorbic acid, acorbyl palmitate, butylated        hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid,        monothioglycerol, propyl gallate, sodium formaldehyde        sulfoxylate, sodium metabisulfite, sodium thiosulfate, sulfur        dioxide, tocopherol, tocopherols excipient;    -   Buffering agents, like acetic acid, ammonium carbonate, ammonium        phosphate, boric acid, citric acid, lactic acid, phosphoric        acid, potassium citrate, potassium metaphosphate, potassium        phosphate monobasic, sodium acetate, sodium citrate, sodium        lactate solution, dibasic sodium phosphate, monobasic sodium        phosphate, histidine.    -   Chelating agents, like edetate disodium,        ethylenediaminetetraacetic acid and salts, edetic acid;    -   Coating agents, like sodium carboxymethylcellulose, cellulose        acetate, cellulose acetate phthalate, ethylcellulose, gelatin,        pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl        methylcellulose, hydroxypropyl methylcellulose phthalate,        methacrylic acid copolymer, methylcellulose, polyethylene        glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium        dioxide, carnauba wax, microcrystalline wax, zein, poly amino        acids, other polymers like PLGA etc., and SAIB.    -   Coloring agent, like ferric oxide.    -   Complexing agents, like ethylenediaminetetraacetic acid and        salts (EDTA), edetic acid, gentisic acid ethanolamide,        oxyquinoline sulfate.    -   Desiccants, like calcium chloride, calcium sulfate, silicon        dioxide.    -   Emulsifying and/or solubilizing agents, like acacia,        cholesterol, diethanolamine (adjunct), glyceryl monostearate,        lanolin alcohols, lecithin, mono- and di-glycerides,        monoethanolamine (adjunct), oleic acid (adjunct), oleyl alcohol        (stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxyl        35 caster oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 10        oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40        stearate, polysorbate 20, polysorbate 40, polysorbate 60,        polysorbate 80, propylene glycol diacetate, propylene glycol        monostearate, sodium lauryl sulfate, sodium stearate, sorbitan        monolaurate, soritan monooleate, sorbitan monopalmitate,        sorbitan monostearate, stearic acid, trolamine, emulsifying wax.    -   Filtering aids, like powdered cellulose, purified siliceous        earth.    -   Flavors and perfumes, like anethole, benzaldehyde, ethyl        vanillin, menthol, methyl salicylate, monosodium glutamate,        orange flower oil, peppermint, peppermint oil, peppermint        spirit, rose oil, stronger rose water, thymol, tolu balsam        tincture, vanilla, vanilla tincture, vanillin.    -   Glidant and/or anticaking agents, like calcium silicate,        magnesium silicate, colloidal silicon dioxide, talc.    -   Humectants, like glycerin, hexylene glycol, propylene glycol,        sorbitol;    -   Ointment bases, like lanolin, anhydrous lanolin, hydrophilic        ointment, white ointment, yellow ointment, polyethylene glycol        ointment, petrolatum, hydrophilic petrolatum, white petrolatum,        rose water ointment, squalane.    -   Plasticizers, like castor oil, lanolin, mineral oil, petrolatum,        benzyl benyl formate, chlorobutanol, diethyl pthalate, sorbitol,        diacetylated monoglycerides, diethyl phthalate, glycerin,        glycerol, mono- and di-acetylated monoglycerides, polyethylene        glycol, propylene glycol, triacetin, triethyl citrate, ethanol.    -   Polypeptides, like low molecular weight (less than about 10        residues);        Proteins, such as serum albumin, gelatin, or immunoglobulins;    -   Polymer membranes, like cellulose acetate membranes.    -   Solvents, like acetone, alcohol, diluted alcohol, amylene        hydrate, benzyl benzoate, butyl alcohol, carbon tetrachloride,        chloroform, corn oil, cottonseed oil, ethyl acetate, glycerin,        hexylene glycol, isopropyl alcohol, methyl alcohol, methylene        chloride, methyl isobutyl ketone, mineral oil, peanut oil,        polyethylene glycol, propylene carbonate, propylene glycol,        sesame oil, water for injection, sterile water for injection,        sterile water for irrigation, purified water, liquid        triglycerides, liquid waxes, higher alcohols.    -   Sorbents, like powdered cellulose, charcoal, purified siliceous        earth, Carbon dioxide sorbents, barium hydroxide lime, soda        lime.    -   Stiffening agents, like hydrogenated castor oil, cetostearyl        alcohol, cetyl alcohol, cetyl esters wax, hard fat, paraffin,        polyethylene excipient, stearyl alcohol, emulsifying wax, white        wax, yellow wax.    -   Suppository bases, like cocoa butter, hard fat, polyethylene        glycol;    -   Suspending and/or viscosity-increasing agents, like acacia,        agar, alginic acid, aluminum monostearate, bentonite, purified        bentonite, magma bentonite, carbomer 934p,        carboxymethylcellulose calcium, carboxymethylcellulose sodium,        carboxymethycellulose sodium 12, carrageenan, microcrystalline        and carboxymethylcellulose sodium cellulose, dextrin, gelatin,        guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose,        hydroxypropyl methylcellulose, magnesium aluminum silicate,        methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol,        povidone, propylene glycol alginate, silicon dioxide, colloidal        silicon dioxide, sodium alginate, tragacanth, xanthan gum;    -   Sweetening agents, like aspartame, dextrates, dextrose,        excipient dextrose, fructose, mannitol, saccharin, calcium        saccharin, sodium saccharin, sorbitol, solution sorbitol,        sucrose, compressible sugar, confectioner's sugar, syrup;    -   Tablet binders, like acacia, alginic acid, sodium        carboxymethylcellulose, microcrystalline cellulose, dextrin,        ethylcellulose, gelatin, liquid glucose, guar gum, hydroxypropyl        methylcellulose, methycellulose, polyethylene oxide, povidone,        pregelatinized starch, syrup.    -   Tablet and/or capsule diluents, like calcium carbonate, dibasic        calcium phosphate, tribasic calcium phosphate, calcium sulfate,        microcrystalline cellulose, powdered cellulose, dextrates,        dextrin, dextrose excipient, fructose, kaolin, lactose,        mannitol, sorbitol, starch, pregelatinized starch, sucrose,        compressible sugar, confectioner's sugar;    -   Tablet disintegrants, like alginic acid, microcrystalline        cellulose, croscarmellose sodium, corspovidone, polacrilin        potassium, sodium starch glycolate, starch, pregelatinized        starch.    -   Tablet and/or capsule lubricants, like calcium stearate,        glyceryl behenate, magnesium stearate, light mineral oil,        polyethylene glycol, sodium stearyl fumarate, stearic acid,        purified stearic acid, talc, hydrogenated vegetable oil, zinc        stearate;    -   Tonicity agent, like dextrose, glycerin, mannitol, potassium        chloride, sodium chloride Vehicle: flavored and/or sweetened        aromatic elixir, compound benzaldehyde elixir, iso-alcoholic        elixir, peppermint water, sorbitol solution, syrup, tolu balsam        syrup.    -   Vehicles, like oleaginous almond oil, corn oil, cottonseed oil,        ethyl oleate, isopropyl myristate, isopropyl palmitate, mineral        oil, light mineral oil, myristyl alcohol, octyldodecanol, olive        oil, peanut oil, persic oil, sesame oil, soybean oil, squalane;        solid carrier sugar spheres; sterile bacteriostatic water for        injection, bacteriostatic sodium chloride injection, liquid        triglycerides, liquid waxes, higher alcohols    -   Water repelling agents, like cyclomethicone, dimethicone,        simethicone;    -   Wetting and/or solubilizing agents, like benzalkonium chloride,        benzethonium chloride, cetylpyridinium chloride, docusate        sodium, nonoxynol 9, nonoxynol 10, octoxynol 9, poloxamer,        polyoxyl 35 castor oil, polyoxyl 40, hydrogenated castor oil,        polyoxyl 50 stearate, polyoxyl 10 oleyl ether, polyoxyl 20,        cetostearyl ether, polyoxyl 40 stearate, polysorbate 20,        polysorbate 40, polysorbate 60, polysorbate 80, sodium lauryl        sulfate, sorbitan monolaureate, sorbitan monooleate, sorbitan        monopalmitate, sorbitan monostearate, tyloxapol;

The crystals may be combined with a polymeric carrier to provide forstability and/or sustained release. Such polymers include biocompatibleand biodegradable polymers.

A polymeric carrier may be a single polymer type or it may be composedof a mixture of polymer types. Nonlimiting examples of polymericcarriers have already been stated above.

Examples of preferred ingredients or excipients include:

-   -   salts of amino acids such as glycine, arginine, aspartic acid,        glutamic acid, lysine, asparagine, glutamine, proline,        histidine;    -   monosaccharides, such as glucose, fructose, galactose, mannose,        arabinose, xylose, ribose;    -   disaccharides, such as lactose, trehalose, maltose, sucrose;    -   polysaccharides, such as maltodextrins, dextrans, starch,        glycogen;    -   alditols, such as mannitol, xylitol, lactitol, sorbitol;    -   glucuronic acid, galacturonic acid;    -   cyclodextrins, such as methyl cyclodextrin,        hydroxypropyl-(3-cyclodextrin)    -   inorganic salts, such as sodium chloride, potassium chloride,        magnesium chloride, phosphates of sodium and potassium, boric        acid ammonium carbonate and ammonium phosphate;    -   organic salts, such as acetates, citrate, ascorbate, lactate;    -   emulsifying or solubilizing agents like acacia, diethanolamine,        glyceryl monostearate, lecithin, monoethanolamine, oleic acid,        oleyl alcohol, poloxamer, polysorbates, sodium lauryl sulfate,        stearic acid, sorbitan monolaurate, sorbitan monostearate, and        other sorbitan derivatives, polyoxyl derivatives, wax,        polyoxyethylene derivatives, sorbitan derivatives; and    -   viscosity increasing reagents like, agar, alginic acid and its        salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide,        cellulose and its derivatives propylene carbonate, polyethylene        glycol, hexylene glycol and tyloxapol.

Formulations described herein also comprise an effective amount ofcrystalline antibody. In particular, the formulations of the inventionmay include a “therapeutically effective amount” or a “prophylacticallyeffective amount” of antibody crystals of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A “therapeutically effective amount” of the antibodycrystals may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the antibody toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Suitable dosages can readily be determined using standard methodology.The antibody is suitably administered to the patient at one time or overa series of treatments. Depending on the above mentioned factors, about1 μg/kg to about 50 mg/kg, as for example 0.1-20 mg/kg of antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily or weekly dosage might range from about 1μg/kg to about 20 mg/kg or more, depending on the condition, thetreatment is repeated until a desired suppression of disease symptomsoccurs. However, other dosage regimens may be useful. In some cases,formulations comprise a concentration of antibody of at least about 1g/L or greater when resolubilized. In other embodiments, the antibodyconcentration is at least about 1 g/L to about 100 g/L whenresolubilized.

Crystals of an antibody, or formulations comprising such crystals, maybe administered alone or as part of a pharmaceutical preparation. Theymay be administered by parenteral, oral or topical routes. For example,they may be administered by oral, pulmonary, nasal, aural, anal, dermal,ocular, intravenous, intramuscular, intraarterial, intraperitoneal,mucosal, sublingual, subcutaneous, transdermal, topical or intracranialroutes, or into the buccal cavity. Specific examples of administrationtechniques comprise pulmonary inhalation, intralesional application,needle injection, dry powder inhalation, skin electroporation, aerosoldelivery, and needle-free injection technologies, including needle-freesubcutaneous administration.

The present invention will now be explained in more detail by means ofthe following, non-limiting, illustrative examples. Guided by thegeneral part of the description and on the basis of his generalknowledge a skilled reader will be enabled to provide furtherembodiments to the invention without undue experimentation.

Experimental Part A. Materials a) Protein

Frozen monoclonal antibody (mAb) D2E7 was obtained from AbbottLaboratories. All experiments were performed from a drug product lotwhere the original mAb concentration was 50 mg/ml.

b) Fine Chemicals

Sodium acetate was obtained from Grüssing GmbH, Filsum.Polyethyleneglycols of different polymerization grades were obtainedfrom Clariant GmbH, Sulzbach. Furthermore, commercial crystallizationscreens and reagents (Hampton Research, Nextal Biotechnologies) wereused for certain microscale experiments. All other chemicals were fromSigma-Aldrich, Steinheim, or Merck, Darmstadt.

B. General Methods a) Thawing of D2E7 Drug Substance

D2E7 was thawed at 25° C. in agitated water baths.

b) Buffer Exchange—Method A

An aliquot of D2E7 solution was pipetted into a 30 KDa MWCO Vivaspin 20concentrator (Vivascience). The protein sample was diluted with the newbuffer in a ratio of 1:10, and by centrifugation at 5,000×g at 4° C.(Sigma 4 K 15 lab centrifuge) the sample volume was brought back to theoriginal sample volume. The dilution/centrifugation steps were repeatedonce, resulting in a dilution of 1:100 of the original sample buffer.After adjustment of protein concentration, the solution was sterilefiltered through a 0.2 μm syringe driven filter unit.

b) Buffer Exchange—Method B

An aliquot of D2E7 solution was placed into a SLIDE-A-LYZER dialysiscassette (Pierce Biotechnology Inc.). The dialysis cassette was placedinto a beaker containing the buffer of choice, and the buffer exchangewas performed at 4° C. overnight with stirring. After adjustment ofprotein concentration, the solution was sterile filtered through a 0.2μm syringe driven filter unit.

c) OD280—Protein Concentration Measurements

A ThermoSpectronics UV1 device was used to assess protein concentrationat a wavelength of 280 nm, applying an extinction coefficient of 1.39cm² mg⁻¹. For this purpose, aliquots of crystallization slurries werecentrifuged at 14,000 rpm, and residual protein concentration wasdetermined in the supernatant.

d) pH Measurements

pH measurements were conducted by using a Mettler Toledo MP220 pH meter.Inlab 413 electrodes and Inlab 423 microelectrodes were utilized.

e) Crystallization Methods

e1) Microscale Crystallization—Sitting Drop Vapor Diffusion Hydra II

Initial crystallization screens were performed using a Hydra IIcrystallization robot and Greiner 96 well plates (three drop wells,Hampton Research). After setting up the plates, the wells were sealedwith Clearseal film (Hampton Research).

e2) Microscale Crystallization—Hanging Drop Vapor Diffusion

Hanging drop vapor diffusion experiments were conducted using VDX plates(with sealant, Hampton Research) and OptiClear plastic cover slides(squares, Hampton Research) or siliconized glass cover slides (circle,Hampton Research), respectively. After preparation of reservoirsolutions, one drop of reservoir solution was admixed with one drop ofthe protein solution on a cover slide, and the well was sealed with theinverted cover slide in such a way that the drop was hanging above thereservoir.

e3) Batch Crystallization—Method A (24 Well Plate)

Batch crystallization was performed by admixing the protein solutionwith an equal amount of crystallization buffer (500 μl) in a well. Thewell was subsequently sealed with adhesive tape to prevent waterevaporation.

e4) Batch Crystallization—Method B (Eppendorff Reaction Tube)

Batch crystallization was performed by admixing the protein solutionwith an equal amount of crystallization buffer in a 1.5 mL or a 2 mLEppendorff reaction tube.

e5) Batch Crystallization—Method C (Falcon Tubes, Agitation)

Batch crystallization was performed by admixing the protein solutionwith an equal amount of crystallization buffer in a 50 mL Falcon tube.Right after closing, the tube was put on a laboratory shaker (GFL 3013or GFL 3015) or was alternatively agitated by tumbling. By applicationof these methods, introduction of stirrers into the sample was avoided.

e6) Batch Crystallization—Method D (1 Liter Polypropylene Container)

Batch crystallization was performed by admixing the protein solutionwith an equal amount of crystallization buffer in a sterilized 1 literpolypropylene bottle. Right after closing, the container was stored atambient temperature without agitation. By application of this method,introduction of stirrers into the sample was avoided.

f) SDS-PAGE

Samples were prepared by adjusting protein concentration to 8 μg/20 μL.The samples were diluted with an SDS/Tris/Glycerine buffer containingbromphenolblue.

Qualitative SDS PAGE analysis was performed using Invitrogen NuPage 10%Bis-Tris Gels, NuPage MES SDS Running Buffer and Mark12 Wide RangeProtein Standards. 20 μL of sample was pipetted into a gel pocket. Afterrunning the gel and fixation with acetic acid/methanol reagent, stainingwas performed using the Novex Colloidal Blue Stain Kit. Gels were driedusing Invitogen Gel-Dry drying solution.

g) Light Microscopy

Crystals were observed using a Zeiss Axiovert 25 or a Nikon Labophotmicroscope. The latter was equipped with a polarization filter set and aJVC TK C1380 color video camera.

h) SE-HPLC

Aggregation levels of D2E7 samples were assessed by SE-HPLC. A DionexP680 pump, ASI-100 autosampler and UVD170U detector device were used.Aggregated species were separated from the monomer by an AmershamBioscience Superose 6 10/300 GL gel filtration column, applying avalidated Abbott standard protocol (CL16-PS-02, Adalimumab purity).

C. Vapor Diffusion Crystallization Experiments

Concentration values given in the following examples are initial valuesreferring to the antibody solution and the reservoir solution beforemixing of the two solutions.

All pH values, if not described otherwise, refer to the pH of an acetatebuffer stock before it was combined with other substances, like thecrystallization agent.

All buffer molarities, if not described otherwise, refer to sodiumacetate concentrations in a stock solution before pH adjustment,typically performed using acetic acid glacial.

Example 1 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water (fullydesalted and optionally pre-destilled) in each well. In this example,the acetate buffer molarity was kept constant at around 0.1 M, and PEG4,000 was varied from around 6% w/v to around 28% w/v in 2% steps. ThepH was around 5.2 throughout. Each condition was assessed in duplicate.Around 1 μL of protein solution was admixed with around 1 μL of aparticular reservoir solution on a square OptiClear plastic cover slide,and the well was sealed with the inverted slide, generating a hangingdrop experiment. The plates were stored at ambient temperature.Microscopy of the drops was performed multiple times during thefollowing thirty days. The conditions were classified into clear drops,drops containing random precipitation, drops containing crystals anddrops containing mixtures of precipitated species and crystals.

Results:

From the 24 wells assessed, no crystals were observed.

Example 2 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 50mg/mL. Except for the protein concentration the process conditions wereidentical with those of Example 1.

Results:

From the 24 wells assessed, no crystals were observed.

Example 3 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG solution and Milli Q water in each well. Inthis example, the acetate buffer molarity was kept constant at around0.1 M, and PEG 400 was varied from around 30% w/v to around 40% w/v in2% steps. The pH was around 5.2 throughout. Each condition was assessedin duplicate. Around 1 μL of protein solution was admixed with around 1μL of a particular reservoir solution on a square OptiClear plasticcover slide, and the well was sealed with the inverted slide, generatinga hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following thirty days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

Results:

From the 12 wells assessed, no crystals were observed.

Example 4 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 50mg/mL. Except for the protein concentration the process conditions wereidentical with those of Example 3.

Results:

From the 12 wells assessed, no crystals were observed.

Example 5 PEG 10,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG solution and Milli Q water in each well. Inthis example, the acetate buffer molarity was kept constant at around0.1 M, and PEG 10,000 was varied from around 4% w/v to around 14% w/v in2% steps. The pH was around 5.2 throughout. Each condition was assessedin duplicate. Around 1 μL of protein solution was admixed with around 1μL of a particular reservoir solution on a square OptiClear plasticcover slide, and the well was sealed with the inverted slide, generatinga hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following thirty days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

Results:

From the 12 wells assessed, no crystals were observed.

Example 6 PEG 10,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 45 to55 mg/mL, preferably 50 mg/mL. Except for the protein concentration theprocess conditions were identical with those of Example 5.

Results:

From the 12 wells assessed, no crystals were observed.

Example 7 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 10,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and PEG 400 was around 32% w/v and around 34% w/v. The pHwas around 4.2, 4.7, 5.2, 5.7, 6.2 or 6.7. Each condition was assessedin duplicate. Around 1 μL of protein solution was admixed with around 1μL of a particular reservoir solution on a square OptiClear plasticcover slide, and the well was sealed with the inverted slide, generatinga hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following thirty days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

Results:

From the 24 wells assessed, no crystals were observed.

Example 8 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration and Set Up

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 50mg/mL. Except for the protein concentration the process conditions wereidentical with those of Example 7.

Results:

From the 24 wells assessed, no crystals were observed.

Example 9 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG solution and Milli Q water in each well. Inthis example, the acetate buffer molarity was used at around 0.025 M,0.05 M, 0.075 M, 0.15 M, 0.2 M or 0.25 M. PEG 400 was varied from around32% w/v to around 34% w/v. The pH was around 5.7 or 4.2. Each conditionwas assessed in duplicate. Around 1 μL of protein solution was admixedwith around 1 μL of a particular reservoir solution on a squareOptiClear plastic cover slide, and the well was sealed with the invertedslide, generating a hanging drop experiment. The plates were stored atambient temperature. Microscopy of the drops was performed multipletimes during the following thirty days. The conditions were classifiedinto clear drops, drops containing random precipitation, dropscontaining crystals and drops containing mixtures of precipitatedspecies and crystals.

Results:

From the 48 wells assessed, no crystals were observed.

Example 10 PEG 400/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 400 solution and Milli Q water in each well.In this example, the acetate buffer molarity was used at around 0.025 M,0.05 M or 0.1 M. PEG 400 was around 28% w/v or around 30% w/v. The pHwas around 5.2, 5.7, 6.2 or 6.7. Each condition was assessed induplicate. Around 1 μL of protein solution was admixed with around 1 μLof a particular reservoir solution on a square OptiClear plastic coverslide, and the well was sealed with the inverted slide, generating ahanging drop experiment. The plates were stored at ambient temperature.Microscopy of the drops was performed multiple times during thefollowing thirty days. The conditions were classified into clear drops,drops containing random precipitation, drops containing crystals anddrops containing mixtures of precipitated species and crystals.

Results:

From the 48 wells assessed, no crystals were observed.

Example 11 PEG 400 Combined with PEG 4,000/Sodium Acetate Grid Screen inHanging Drop Vapor Diffusion Mode

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and PEG 4,000 was varied from around 4% w/v to around 8%w/v in 2% steps. Simultaneously, PEG 400 was added to the PEG4,000/acetate solutions at concentrations of around 24% w/v, 26% w/v,28% w/v or 30% w/v. The pH was around 5.2 throughout. Each condition wasassessed in duplicate. Around 1 μL of protein solution was admixed witharound 1 μL of a particular reservoir solution on a square OptiClearplastic cover slide, and the well was sealed with the inverted slide,generating a hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following thirty days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

Results:

From the 24 wells assessed, no crystals were observed.

Example 12 PEG 400 Combined with PEG 4,000/Sodium Acetate Grid Screen inHanging Drop Vapor Diffusion Mode, Different Set Up

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.2. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and PEG 4,000 was varied from around 4% w/v to around 8%w/v in 2% steps. Simultaneously, PEG 400 was added to the PEG4,000/acetate solutions at concentrations of around 30% w/v, 32% w/v,34% w/v or 36% w/v. The pH was around 4.2 throughout. Each condition wasassessed in duplicate. Around 1 μL of protein solution was admixed witharound 1 μL of a particular reservoir solution on a square OptiClearplastic cover slide, and the well was sealed with the inverted slide,generating a hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following thirty days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

Results:

From the 24 wells assessed, no crystals were observed.

Example 13 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0. The proteinconcentration was adjusted to 10 mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and PEG 4,000 was varied from around 4% w/v to around 26%w/v in 2% steps. The pH of the acetate buffer used was the same as thecorresponding protein buffer. Each condition was assessed in duplicate.Around 1 μL of protein solution was admixed with around 1 μL of aparticular reservoir solution on a square OptiClear plastic cover slide,and the well was sealed with the inverted slide, generating a hangingdrop experiment. The plates were stored at ambient temperature.Microscopy of the drops was performed multiple times during thefollowing thirty days. The conditions were classified into clear drops,drops containing random precipitation, drops containing crystals anddrops containing mixtures of precipitated species and crystals.

Results:

From the 144 wells assessed, no crystals were observed.

Example 14 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

The experimental conditions were identical to Example 13, except for theacetate buffer molarity, which was kept constant at around 0.2 M(molarity of precipitation buffer).

Results:

From the 144 wells assessed, no crystals were observed.

Example 15 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

The experimental conditions were identical to Example 13, except for theacetate buffer molarity which was kept constant at 0.1 M (molarity ofprecipitation buffer).

Results:

From the 144 wells assessed, no crystals were observed.

Example 16 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

The experimental conditions were identical to Example 13, except for theacetate buffer molarity which was kept constant at around 0.4 M(molarity of precipitation buffer).

Results:

From the 144 wells assessed, no crystals were observed.

Example 17 PEG 4,000/Sodium Acetate Bulk Experiments

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.5. The protein concentration was adjusted to 10mg/mL.

Four aliquots of 500 μL each were pipetted into four Eppendorff reactiontubes. A 24% PEG 4,000 in 0.1 M sodium acetate buffer at pH 5.5 solutionwas titrated to the protein solutions until the solution became slightlyopaque. Subsequently, water was pipetted to the solutions just until thesolutions became clear again. This method is referred to as bulkcrystallization. Titration was performed at ambient temperature for twosamples and at 4° C. for the two other samples. Subsequently, one ofeach pair of samples was stored at ambient temperature or at 4° C.,respectively. Microscopy of 1 μL aliquots of the samples was performedmultiple times during the following week

Results:

From the four samples, none rendered crystals.

Example 18 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Temperature

The experimental conditions were identical to Example 13. However, thetubes were set up and stored at 4° C.

Results:

From the 144 wells assessed, no crystals were observed.

Example 19 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Protein Concentration

Except for the protein concentration, which was adjusted to 5 mg/mL theexperimental conditions were identical to Example 13.

Results:

From the 144 wells assessed, no crystals were observed.

Example 20 Ammonium Sulfate/Sodium Acetate Grid Screen in Hanging DropVapor Diffusion Mode

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.5. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, ammonium sulfate stock solution and Milli Q water ineach well. In this example, the acetate buffer molarity was keptconstant at around 0.1 M, and the ammonium sulfate concentration wasvaried from 0.5 M to 2.5 M in steps of 0.25 M. The pH of the acetatebuffer was around 5.5 throughout. Each condition was assessed induplicate. Around 1 μL of protein solution was admixed with around 1 μLof a particular reservoir solution on a square OptiClear plastic coverslide, and the well was sealed with the inverted slide, generating ahanging drop experiment. The plates were set up and stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following two weeks. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

Results:

From the 18 wells assessed, no crystals were observed.

Example 21 Sodium Chloride/Sodium Acetate Grid Screen in Hanging DropVapor Diffusion Mode

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.5. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, sodium chloride stock solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and the sodium chloride concentration was varied from 1.5M to 2.5 M, varied in steps of 0.5 M. The pH of the acetate buffer wasaround 5.5 throughout. Each condition was assessed in duplicate. Around1 μL of protein solution was admixed with around 1 μL of a particularreservoir solution on a square OptiClear plastic cover slide, and thewell was sealed with the inverted slide, generating a hanging dropexperiment. The plates were set up and stored at ambient temperature.Microscopy of the drops was performed multiple times during thefollowing two weeks. The conditions were classified into clear drops,drops containing random precipitation, drops containing crystals anddrops containing mixtures of precipitated species and crystals.

Results:

From the 6 wells assessed, no crystals were observed.

Example 22 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Influence of Detergents

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.5. The protein concentration was adjusted to 5mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and PEG 4,000 was varied from around 10% w/v to around 20%w/v in 2% steps. The pH of the acetate buffer was around 5.5 throughout.Furthermore, polysorbate 20, polysorbate 80 and Pluronic F 68 were addedto any resulting buffer as described above at concentrations of 0%,0.02% and 0.1%, respectively. Around 1 μL of protein solution wasadmixed with around 1 μL of a particular reservoir solution on a squareOptiClear plastic cover slide, and the well was sealed with the invertedslide, generating a hanging drop experiment. The plates were set up andstored at ambient temperature. Microscopy of the drops was performedmultiple times during the following two weeks. The conditions wereclassified into clear drops, drops containing random precipitation,drops containing crystals and drops containing mixtures of precipitatedspecies and crystals.

Results:

From the 84 wells assessed, no crystals were observed. No influence ofthe assessed detergents on the behaviour of the crystallization systemcould be observed.

Example 23 Zinc Acetate/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode

D2E7 was buffered into a buffer containing around 0.1 M sodium acetateat a pH of around 5.5. The protein concentration was adjusted to 10mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, zinc acetate stock solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and the zinc acetate concentration was varied from 0.1 Mto 0.9 M in steps of around 0.2 M. The pH of the acetate buffer wasaround 5.5 throughout. Each condition was assessed in duplicate. Around1 μL of protein solution was admixed with around 1 μL of a particularreservoir solution on a square OptiClear plastic cover slide, and thewell was sealed with the inverted slide, generating a hanging dropexperiment. The plates were set up and stored at ambient temperature.Microscopy of the drops was performed multiple times during thefollowing two weeks. The conditions were classified into clear drops,drops containing random precipitation, drops containing crystals anddrops containing mixtures of precipitated species and crystals.

Results:

From the 12 wells assessed, no crystals were observed.

Example 24 Broad Screening of Conditions in Vapor Diffusion Mode

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL, 10 mg/mL, or 20mg/mL.

Using the Hydra II crystallization robot, 96 well Greiner plates wereset up at ambient temperature, using several commercially availablecrystallization screens. The protein solution and the crystallizationagent were admixed in a ratio of around 1:1, preferably 1:1.

The following screens were used:

Hampton Crystal Screen 1 & 2 (Hampton Research), Hampton Index Screen(Hampton Research), Hampton SaltRX Screen (Hampton Research),

Nextal The Classics, The Classics Lite, The PEGs, The Anions, The pHclear and The Ammonium sulfate (all from Nextal Biotechnologies).

After addition of protein to the crystallization agent (three drops percondition, containing the three different protein concentrations asdescribed above), the plates were sealed with Clearseal film. Each platewas set up in quadruplicate and then stored at ambient temperature, 4°C., 27° C. and 37° C., respectively. Microscopy of the drops wasperformed after five days and twelve days, respectively. The conditionswere classified into clear drops, drops containing random precipitation,drops containing crystals and drops containing mixtures of precipitatedspecies and crystals.

Results:

From the 864 commercial conditions evaluated, 2 rendered crystals atprotein concentrations and temperatures as defined below, at least aftertwo weeks.

-   -   0.1 M sodium acetate anhydrous pH 4.6, 0.9 M sodium dihydrogen        phosphate, 0.9 M potassium dihydrogen phosphate (=Nextal The        Anions, E3), 10 or 20 mg/mL, and 27° C., or 20 mg/mL and 37° C.    -   0.1 M Bis-Tris Propane pH 7.0, 1.5 M ammonium sulfate (=Hampton        SaltRX, F2), 5, 10, or 20 mg/mL. and 27° C.

The crystals showed needle cluster-like morphologies.

The following conditions from commercially available screens did notrender crystals. For detailed solution compositions, please refer towww.hamptonresearch.com and www.nextalbiotech.com:

Hampton Crystal Screen 1—all conditions (48)Hampton Crystal Screen 2—all conditions (48)Hampton Index Screen—all conditions (96)Hampton SaltRX Screen—all conditions despite “F2” (95)Nextal—The Classics—all conditions (96)Nextal—The Classics Lite—all conditions (96)Nextal—The PEGs—all conditions (96)Nextal—The Anions—all conditions despite “E3” (95)Nextal—The pH Clear—all conditions (96)Nextal—The AmmoniumSulfate—all conditions (96)

Example 25 PEG 4,000/Sodium Acetate Grid Screen in Hanging Drop VaporDiffusion Mode, Different Set Up

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL, 10 mg/mL, or 20mg/mL.

A greased VDX plate and circle siliconized glass cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and PEG 4,000 concentration was varied from 4% to 26% in2% steps. The pH was around 5.5 throughout. Each condition was set upwith the three protein concentrations as described above. Around 1 μL ofprotein solution was admixed with around 1 μL of a particular reservoirsolution on a circle siliconized glass cover slide, and the well wassealed with the inverted slide, generating a hanging drop experiment.The plates were stored at ambient temperature. Microscopy of the dropswas performed after six days. The conditions were classified into cleardrops, drops containing random precipitation, drops containing crystalsand drops containing mixtures of precipitated species and crystals.

Results:

From the 72 wells assessed, no crystals were observed.

Example 26 Hanging Drop Vapor Diffusion Experiments Applying the HamptonDetergent Screen

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL.

A greased VDX plate and circle siliconized glass cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, 50% w/v PEG 4,000 solution and Milli Q water in eachwell. In this example, the acetate buffer molarity was kept constant ataround 0.1 M, and the PEG 4,000 concentration around 12% w/v or 14% w/v.The pH was around 5.5 throughout. Around 4 μL of protein solution wasadmixed with around 1 μL of a particular detergent solution of theHampton screen on a circle siliconized glass cover slide. The drop wassubsequently admixed with 5 μL of a particular reservoir solution, andthe well was sealed with the inverted slide, generating a hanging dropexperiment. The plates were stored at ambient temperature. Microscopy ofthe drops was performed after six days. The conditions were classifiedinto clear drops, drops containing random precipitation, dropscontaining crystals and drops containing mixtures of precipitatedspecies and crystals.

Results:

From the 144 wells assessed, no crystals were observed.

Example 27 Hanging Drop Vapor Diffusion Using Hampton PEG/Ion Screen

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL or 10 mg/mL.

Greased VDX plates and circle siliconized glass cover slides were used.500 μL of each of the 48 buffer formulations was pipetted into a welland admixed with 250 μL of Milli Q water, respectively. Around 1 μL ofprotein sample was pipetted onto a cover slide and subsequently admixedwith around 1 μL of the reservoir solution of a particular well. Thewell was sealed with the inverted cover slide, generating a hanging dropexperiment. The plates were stored at ambient temperature. Microscopy ofthe drops was performed multiple times during the following seven days.The conditions were classified into clear drops, drops containing randomprecipitation, drops containing crystals and drops containing mixturesof precipitated species and crystals.

Results:

From the 96 conditions tested, no crystals were observed.

Example 28 Hanging Drop Vapor Diffusion Using Hampton PEG/Ion Screen,Different Set Up

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL.

The experimental conditions were identical with those of Example 27 withthe exception that 500 μL of each of the 48 buffer formulations waspipetted into a well and admixed with 500 μL of Milli Q water,respectively.

Results:

From the 48 conditions tested, no crystals were observed.

Example 29 Hanging Drop Vapor Diffusion Using Hampton Low Ionic StrengthScreen

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL.

Greased VDX plates and circle siliconized glass cover slides were used.1 mL of 24% w/v PEG 3,350 dehydrant solution was pipetted into 108wells, respectively. Around 2 μL of protein sample were pipetted onto acover slide and subsequently admixed with around 1 μL of one of the 18particular buffer reagents. Thereafter, around 2.5 μL of PEG 3,350precipitant of one of six different concentrations was added to thedrop. The wells were sealed with the inverted cover slides, generating108 different hanging drop experiments.

The plates were stored at ambient temperature. Microscopy of the dropswas performed multiple times during the following seven days. Theconditions were classified into clear drops, drops containing randomprecipitation, drops containing crystals and drops containing mixturesof precipitated species and crystals.

Results:

From the 108 conditions tested, none rendered crystals.

Example 30 Ammonium Sulfate/Bis-Tris Propane Grid Screen in Hanging DropVapor Diffusion Mode

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL, 10 mg/mL, or 20mg/mL.

A greased VDX plate and circle siliconized glass cover slides were used.500 μL of a particular reservoir solution was prepared by admixingammonium sulfate stock solution, Bis-Tris propane stock solution andMilli Q water in each well. In this example, ammonium sulfate molaritywas around 0.5 M, 1 M, 1.5 M or 2 M. The Bis-Tris Propane molarity was0.1 M throughout, and the Bis-Tris Propane buffer pH was around 5.5,6.0, 6.5, 7.0, 7.5 or 8.0. The resulting 24 conditions were assessedwith all of the three protein concentrations as described above, andwith storage at ambient temperature or storage at around 27° C.,respectively. Around 1 μL of protein solution was admixed with around 1μL of a particular reservoir solution on a circle siliconized glasscover slide, and the well was sealed with the inverted slide, generatinga hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed after three days. Theconditions were classified into clear drops, drops containing randomprecipitation, drops containing crystals and drops containing mixturesof precipitated species and crystals.

Results:

From the 144 conditions tested, none rendered crystals after three days.

Example 31 Sodium Potassium Dihydrogen Phosphate/Sodium Acetate GridScreen in Hanging Drop Vapor Diffusion Mode

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL, 10 mg/mL, or 20mg/mL.

A greased VDX plate and square OptiClear plastic cover slides were used.500 μL of a particular reservoir solution was prepared by admixingacetate buffer, sodium dihydrogen phosphate stock solution, potassiumdihydrogen phosphate stock solution and Milli Q water in each well. Inthis example, the acetate buffer molarity was kept constant at around0.1 M, and the acetate buffer pH was around 4.1, 4.6, 5.1 or 5.6.

The following combinations of sodium dihydrogen phosphate and potassiumdihydrogen phosphate were applied:

-   -   around 0.3 M sodium dihydrogen phosphate and around 0.3 M        potassium dihydrogen phosphate;    -   around 0.6 M sodium dihydrogen phosphate and around 0.6 M        potassium dihydrogen phosphate;    -   around 0.9 M sodium dihydrogen phosphate and around 0.9 M        potassium dihydrogen phosphate;    -   around 1.8 M sodium dihydrogen phosphate,    -   around 2.1 M sodium dihydrogen phosphate,    -   around 2.4 M sodium dihydrogen phosphate.

Each condition was set up with the three protein concentrations asdescribed above. Around 1 μL of protein solution was admixed with around1 μL of a particular reservoir solution on a square OptiClear plasticcover slide, and the well was sealed with the inverted slide, generatinga hanging drop experiment. The plates were stored at ambienttemperature. Microscopy of the drops was performed multiple times duringthe following month. The conditions were classified into clear drops,drops containing random precipitation, drops containing crystals anddrops containing mixtures of precipitated species and crystals.

Results:

From the 72 wells assessed, the following crystallization buffersgenerated crystals in the shape of needle clusters:

-   -   around 0.9 M sodium dihydrogen phosphate and around 0.9 M        potassium dihydrogen phosphate, at pH around 4.1;    -   around 1.8 M sodium dihydrogen phosphate without the potassium        salt, at pH around 4.6.

Crystals were obtained with these conditions at all three proteinconcentrations.

Example 32 Sodium Potassium Dihydrogen Phosphate/Sodium Acetate GridScreen in Hanging Drop Vapor Diffusion Mode, Different Temperature

The experimental conditions were identical with those of Example 31,except that the storage temperature was increased to 30° C.

Results:

From the 72 wells assessed, following crystallization buffers generatedcrystals in the shape of needle clusters:

Protein concentration of around 5 mg/mL:

-   -   around 0.9 M sodium dihydrogen phosphate and around 0.9 M        potassium dihydrogen phosphate at pH around 4.1;    -   around 1.8 M sodium dihydrogen phosphate without the potassium        salt, at pH around 4.1.    -   around 1.8 M sodium dihydrogen phosphate without the potassium        salt, at pH around 4.6.

Around 1.8 M sodium dihydrogen phosphate without the potassium salt, atpH around 5.1.

Protein concentration of around 10 mg/mL:

-   -   around 0.9 M sodium dihydrogen phosphate and around 0.9 M        potassium dihydrogen phosphate, at pH around 4.1.    -   around 1.8 M sodium dihydrogen phosphate without the potassium        salt, at pH

around 4.6.

-   -   around 1.8 M sodium dihydrogen phosphate without the potassium        salt, at pH around 5.1.

Protein concentration of around 20 mg/mL:

-   -   around 0.9 M sodium dihydrogen phosphate and around 0.9 M        potassium dihydrogen phosphate at pH around 4.1 and    -   around 1.8 M sodium dihydrogen phosphate without the potassium        salt, at pH around 4.1.

Discussion of Results of Vapor Diffusion Crystallization Experiments:

Crystallization experiments were initially performed using awell-described micro scale methodology. Since a PEG 4,000/sodium acetatebuffer condition was described as a promising crystallization conditionby other inventors who were working with different antibodies ofdifferent antigen specificity or origin, it was decided to start withthese agents. It was found after extensive experimentation that PEG4,000 in an acetate buffer did not provide crystals, at least atinvestigated combinations of factors influencing crystallization(protein concentration, precipitating agent concentration, buffer ionicstrength and pH, temperature), and thus it was decided to continue withbroad crystallization screens, thereby introducing a wide variety ofchemicals into the screening process. Finally, it was surprisingly foundthat sodium dihydrogen phosphate in acetate buffer is a powerfulcrystallization agent for D2E7, which does not introduce any toxicreagent unacceptable from a pharmaceutical point of view.

D. Batch Crystallization Experiments

Concentration values given in the following examples are initial valuesreferring to the antibody solution and the crystallization solutionbefore mixing of the two solutions.

All pH values, if not described otherwise, refer to the pH of an acetatebuffer stock before it was combined with other substances, like thecrystallization agent.

All buffer molarities, if not described otherwise, refer to sodiumacetate concentrations in a stock solution before pH adjustment,typically performed using acetic acid glacial.

Example 33 Sodium Potassium Dihydrogen Phosphate/Sodium Acetate BatchCrystallization at 800 μL Batch Volume

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 5 mg/mL, 10 mg/mL, or 20mg/mL.

Batch crystallization was performed by admixing around 400 μL of eachprotein solution with an equal amount of crystallization solution in a1.5 mL Eppendorff reaction tube. 400 μL of a particular crystallizationsolution was prepared by admixing acetate buffer, sodium dihydrogenphosphate stock solution, potassium dihydrogen phosphate stock solutionand Milli Q water. In this example, the acetate buffer molarity was 0.1M, and the acetate buffer pH was around 4.1. The following combinationof sodium dihydrogen phosphate and potassium dihydrogen phosphate wasused: around 0.9 M sodium dihydrogen phosphate and around 0.9 Mpotassium dihydrogen phosphate. The reaction tubes were stored atambient temperature. Microscopy of 1 μL aliquots was performed after 11days.

Results:

No crystals were observed after 11 days.

Example 34 Sodium Dihydrogen Phosphate/Sodium Acetate BatchCrystallization at 600 μL Batch Volume

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing around 300 μL of theprotein solution with an equal amount of crystallization solution in a1.5 mL Eppendorff reaction tube. 300 μL of a particular crystallizationsolution was prepared by admixing acetate buffer, sodium dihydrogenphosphate stock solution and Milli Q water. In this example, the acetatebuffer molarity was 0.1 M, and the acetate buffer pH was around 4.1.Sodium dihydrogen phosphate molarity was around 1.5 M, 1.8 M, 2.1 M and2.4 M, respectively. The reaction tubes were stored at ambienttemperature. Microscopy of 1 μL aliquots was performed after 11 days.

Results:

No crystals were observed after 11 days.

Example 35 Sodium Potassium Dihydrogen Phosphate/Sodium Acetate GridScreen Batch Crystallization at 1 mL Batch Volume

D2E7 was used without exchanging the buffer. Thus, the initialcomposition was D2E7 50 mg/mL, mannitol 12 mg/mL, polysorbate 80 1mg/mL, citric acid monohydrate 1.305 mg/mL, sodium citrate 0.305 mg/mL,disodium hydrogen phosphate dihydrate 1.53 mg/mL, sodium dihydrogenphosphate dehydrate 0.86 mg/mL, and sodium chloride 6.16 mg/mL, pH 5.2.

D2E7 was brought to a concentration of around 10 mg/mL by dilution withMilli Q water.

Batch crystallization was performed by admixing around 500 μL of theprotein solution with an equal amount of crystallization solution inwell of a 24 well plate. 500 μL of a particular crystallization solutionwas prepared by admixing acetate buffer, sodium dihydrogen phosphatestock solution, potassium dihydrogen phosphate stock solution and MilliQ water in a well. In this example, the acetate buffer molarity was 0.1M, and the acetate buffer pH was around 4.1, 4.6, 5.1 or 5.6. Thefollowing combinations of sodium dihydrogen phosphate and potassiumdihydrogen phosphate were used:

-   -   around 0.7 M sodium dihydrogen phosphate and around 0.7 M        potassium dihydrogen phosphate,    -   around 0.9 M sodium dihydrogen phosphate and around 0.9 M        potassium dihydrogen phosphate,    -   around 1.8 M sodium dihydrogen phosphate without the potassium        salt,    -   around 2.1 M sodium dihydrogen phosphate without the potassium        salt,    -   around 2.4 M sodium dihydrogen phosphate without the potassium        salt.

The wells were subsequently sealed after preparation of thecrystallization mixture to prevent water evaporation. Microscopy of theplate was performed after 4 days.

Results:

No crystals were observed after 4 days.

Example 36 Sodium Potassium Dihydrogen Phosphate/Sodium Acetate GridScreen Batch Crystallization at 1 mL Batch Volume

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing around 500 μL of theprotein solution with an equal amount of crystallization solution inwell of a 24 well plate. 500 μL of a particular crystallization solutionwas prepared by admixing acetate buffer, sodium dihydrogen phosphatestock solution, potassium dihydrogen phosphate stock solution and MilliQ water in a well. In this example, the acetate buffer molarity was 0.1M, and the acetate buffer pH was around 4.1 or 4.6. The followingcombinations of sodium dihydrogen phosphate and potassium dihydrogenphosphate were applied:

-   -   around 1.8 M sodium dihydrogen phosphate and around 0.8 M        potassium dihydrogen phosphate,    -   around 2.2 M sodium dihydrogen phosphate and around 0.6 M        potassium dihydrogen phosphate,    -   from around 2.6 M sodium dihydrogen phosphate to around 4.4 M        sodium dihydrogen phosphate in 0.2 M steps without the potassium        salt, respectively.

The wells were subsequently sealed after preparation of thecrystallization mixture to prevent water evaporation. Microscopy of theplate was performed multiple times during the following week.Furthermore, the crystal yield of three batches was determined by OD280.An aliquot of the suspension was centrifuged at 14,000 rpm, and theprotein concentration in the supernatant was assessed.

Results:

Needle cluster like crystals were found in the following eight batches:

-   -   acetate buffer pH 4.1 and sodium dihydrogen phosphate molarity        of around 3.6 M to around 4.4 M (in 0.2 M steps),    -   acetate buffer pH 4.6 and sodium dihydrogen phosphate molarity        of around 4.0 M to around 4.4 M (in 0.2 M steps).

Crystal yield was assessed for the batches at acetate buffer pH 4.1 andsodium dihydrogen phosphate molarity of around 4.0 M to around 4.4 M.The crystal yield as determined by OD280 from residual proteinconcentration in the supernatant was above 95% after five days.

Precipitated species were obviously present in these batches immediatelyafter combining the protein solution and the crystallization solution(milky suspension, typical light microscopic picture). As noprecipitated species were observed after five days, it was concludedthat formerly precipitated species rearranged into crystalline species.The protein is highly supersaturated in the crystallization mixture, andprotein precipitates immediately. Some protein may still be dissolved,now either only slightly supersaturated or perhaps even belowsaturation. Crystals form, thereby further lowering the concentration ofdissolved protein. Furthermore, the precipitated species clearlyredissolve over time and are incorporated into the growing crystals.

Example 37 Sodium Dihydrogen Phosphate/Sodium Acetate Grid Screen BatchCrystallization at 1 mL Batch Volume, Different Protein Concentration

D2E7 was used without exchanging the buffer (see Example 35).

D2E7 was brought to a concentration of around 10 mg/mL by diluting theliquid with Milli Q water.

Batch crystallization was performed by admixing around 500 μL of theprotein solution with an equal amount of crystallization solution inwell of a 24 well plate. 500 μL of a particular crystallization solutionwas prepared by admixing acetate buffer, sodium dihydrogen phosphatestock solution, potassium dihydrogen phosphate stock solution and MilliQ water in a well. In this example, the acetate buffer molarity was 0.1M, and the acetate buffer pH was around 4.1 or 4.6. Sodium dihydrogenphosphate molarity was varied from around 2.6 M sodium dihydrogenphosphate to around 4.4 M sodium dihydrogen phosphate in 0.2 M steps.The wells were subsequently sealed after preparation of thecrystallization mixture to prevent water evaporation. Microscopy of theplate was performed multiple times during the following week.Furthermore, the crystal yield of one particular batch was determined byOD280. An aliquot of the suspension was centrifuged at 14,000 rpm, andthe protein concentration in the supernatant was assessed.

Results:

Needle cluster-like crystals were found in the following six batches:

-   -   acetate buffer pH 4.1 and sodium dihydrogen phosphate molarity        of around 3.4 M to around 4.4 M (in 0.2 M steps).

Crystal yield was assessed for the batch at acetate buffer pH 4.1 andsodium dihydrogen phosphate molarity of around 4.2 M. The crystal yieldas determined by OD280 from residual protein concentration in thesupernatant was above 95% after eight days.

Precipitated species were obviously present in these batches immediatelyafter combining the protein solution and the crystallization solution(milky suspension, typical light microscopic picture). As noprecipitated species were observed after six days, it was concluded thata phase transition occurred where formerly precipitated speciesrearranged into crystalline species.

Example 38 Sodium Dihydrogen Phosphate/Sodium Acetate BatchCrystallization at 2 mL Batch Volume

D2E7 was buffered into a 20 mM HEPES/150 mM sodium chloride buffer at pH7.4. The protein concentration was adjusted to 10 mg/mL.

Batch crystallization was performed by admixing around 1 mL of theprotein solution with an equal amount of crystallization solution in a 2mL Eppendorff reaction tube. 1 mL of a particular crystallizationsolution was prepared by admixing acetate buffer, sodium dihydrogenphosphate stock solution and Milli Q water. In this example, the acetatebuffer molarity was 0.1 M, and the acetate buffer pH was around 4.1.Sodium dihydrogen phosphate molarity was around 4.0 M, 4.2 M or 4.4 M.The reaction tubes were stored at ambient temperature. Microscopy of 1μL aliquots was performed multiple times during the following week.

Results:

Needle cluster-like crystals were found in all batches after six days.

Precipitated species were obviously present in these batches immediatelyafter combining the protein solution and the crystallization solution(milky suspension, typical light microscopic picture). Formerlyprecipitated species rearranged into crystalline species as described inExample 36.

Example 39 Sodium Dihydrogen Phosphate/Sodium Acetate Grid Screen BatchCrystallization at 1 mL Batch Volume, Different Protein Concentration

D2E7 was used without exchanging the buffer (see Example 35).

Batch crystallization was performed by admixing around 500 μL of theprotein solution with an equal amount of crystallization solution inwell of a 24 well plate. 500 μL of a particular crystallization solutionwas prepared by admixing acetate buffer, sodium dihydrogen phosphatestock solution, potassium dihydrogen phosphate stock solution and MilliQ water in a well. In this example, the acetate buffer molarity was 0.1M, and the acetate buffer pH was around 4.1. Sodium dihydrogen phosphatemolarity was varied from around 0.2 M to around 4.4 M in 0.2 M steps.The wells were subsequently sealed after preparation of thecrystallization mixture to prevent water evaporation. Microscopy of theplate was performed multiple times during the following week.Furthermore, the crystal yield of the batch was determined by OD280. Analiquot of the suspension was centrifuged at 14,000 rpm, and the proteinconcentration in the supernatant was assessed.

Results:

Needle cluster-like crystals were found in the following two batches:

-   -   sodium dihydrogen phosphate molarity of around 3.4 M and around        3.6 M.

Precipitated species and oily precipitation phases were also present inthese crystal containing batches

Example 40 Sodium Dihydrogen Phosphate/Sodium Acetate BatchCrystallization at 20 mL Batch Volume, Agitation

D2E7 was used without exchanging the buffer (see Example 35).

D2E7 was brought to a concentration of around 10 mg/mL by dilution withMilli Q water.

Batch crystallization was performed by admixing around 10 mL of proteinsolution with an equal amount of crystallization solution in a 50 mLFalcon tube. 10 mL of the crystallization solution was prepared byadmixing acetate buffer, sodium dihydrogen phosphate stock solution andMilli Q water in the tube. In this example, the acetate buffer molaritywas 0.1 M, and the acetate buffer pH was around 4.1. Sodium dihydrogenphosphate molarity was 4.2 M. The tube was stored at ambienttemperature, agitating the batch on a laboratory shaker. Microscopy of a1 μL aliquot of the solution was performed multiple times during thefollowing month.

Results:

Precipitated matter was observed in this batch.

Example 41a Sodium Dihydrogen Phosphate/Sodium Acetate BatchCrystallization at 100 mL Batch Volume, No Agitation

D2E7 was used without exchanging the buffer (see Example 35).

D2E7 was brought to a concentration of around 10 mg/mL by dilution withMilli Q water.

Batch crystallization was performed by admixing around 50 mL of proteinsolution with an equal amount of crystallization solution in a clean 1 Lpolypropylene bottle. 50 mL of the crystallization solution was preparedby admixing acetate buffer, sodium dihydrogen phosphate stock solutionand Milli Q water in the tube. In this example, the acetate buffermolarity was 0.1 M, and the acetate buffer pH was around 4.1. Sodiumdihydrogen phosphate molarity was 4.2 M. The container was stored atambient temperature. Microscopy of a 1 μL aliquot of the solution wasperformed multiple times during the following month.

Results:

Needle cluster like crystals were observed after seven days. The crystalyield as determined by OD280 from residual protein concentration in thesupernatant was above 95% after seven days.

Precipitated species were present in this batch immediately aftercombining the protein solution and the crystallization solution (milkysuspension, typical light microscopic picture). Since no precipitatedspecies were observed after seven days, it was concluded that a phasetransition occurred where formerly precipitated species rearranged intocrystalline species.

Example 41b Sodium Dihydrogen Phosphate/Sodium Acetate BatchCrystallization at 1 mL, 50 mL and 10 L Batch Volume, No Agitation

Large-scale crystallization of D2E7 was also performed by combining 1 Lof 50 mg/mL D2E7 in Adalimumab commercial buffer formulation pH 5.2 (seeExample 35) and 4 L water for injection (WFI) in a 10 L polypropylenevessel (Nalgene®). The solution was homogenized by gentle shaking. This5 L D2E7 solution (10 mg/mL) was then mixed with 5 L of precipitatingagent solution (5 M sodium dihydrogen phosphate, 4,400 mL, 1 M sodiumacetate buffer, pH 4.1, 500 mL, WFI (Ampuwa), 100 mL) The precipitatingagent solution was added in 500 mL portions. After addition of eachportion the solution was homogenized by gently rotating/inverting thebottle. After addition of around 2,500 to −3,000 mL of the precipitatingagent solution, a white precipitate appeared. The remainingprecipitating agent was added all at once. Then the crystal preparationwas homogenized (gently rotating/inverting) again.

Immediately after batch manufacture (i.e. after admixing of 5 L D2E7solution and 5 L precipitating agent), 1 mL (filled into low volumeEppendorf sample vials) and 50 mL aliquots (filled into 50 mL Falconsample tubes) were pulled and stored adjacent to the 10 L vessel forcontrol and for evaluation of the impact of batch size on D2E7crystallization. As outlined by FIGS. 2 to 4, the batch volume (i.e. 1mL, 50 mL and 10 L, respectively) had no impact on D2E7 crystalneedle/needle cluster size.

Discussion of Results of Batch Crystallization Experiments:

As the applied micro scale technique (see Section D. supra) is notfeasible for large scale production of protein crystals, thecrystallization conditions discovered by these micro scale methods weretransferred into a scaleable batch mode.

D2E7 was successfully crystallized at 100 mL batch volume withultimately high yield (>95%) and reproducibility, indicating that thiscrystallization system is applicable for industrial processing. BySDS-PAGE analysis, the protein character of the crystals was proven.SE-HPLC analysis of redissolved crystals showed only a slight increasein aggregated species. Washing of the crystals was possible by using anacetate buffer containing sodium dihydrogen phosphate around 4.2M sodiumdihydrogen phosphate in around 0.1M sodium acetate at a pH around 4.1.No measurable solubility of D2E7 crystals in such a washing bufferoccurs, as analyzed by OD280, recovering more than 90% of the crystals.

The experimental conditions of the above batch experiments aresummarized in the following Table 1:

TABLE 1 Batch Experiments Crystallization Buffer Yield pH pH FinalProtein day of Ex. Batch Volume solution Exchange Crystals Buffer FinalConc. mg/ml Temp. visual control 33 800 μl 0.1M NaAc, NaH2PO4 0.9M, yesnone 4.1 2.5-10 amb 11 d  KH2PO4 0.9M 34 600 μl 0.1M NaAc, NaH2PO41.5-2.4M yes none 4.1 5 amb 11 d  35 1 ml 0.1M NaAc, NaH2PO4 0.7M, none4.1-5.6 5 amb 4 d KH2PO4 0.7M 0.1M NaAc, NaH2PO4 0.9M, 4.1-5.6 KH2PO40.9M 0.1M NaAc, NaH2PO4 1.8M, 4.1-5.6 0.1M NaAc, NaH2PO4 2.1M, 4.1-5.60.1M NaAc, NaH2PO4 2.4M, 4.1-5.6 36 1 ml 0.1M NaAc, NaH2PO4 3.6-4.4Myes >95% 4.1 3.9-3.7 5 amb 5 d 0.1M NaAc, NaH2PO4 4.0-4.4M 4.6 4.0-3.9amb 37 1 ml 0.1M NaAc, NaH2PO4 3.4-4.4M >95% 4.1 3.9-3.7 5 amb 6 d 38 2ml 0.1M NaAc, NaH2PO4 4.0-4.4M yes n.d. 4.1 3.9-3.7 5 amb 6 d 39 1 ml0.1M NaAc, NaH2PO4 3.4-3.6M n.d + 4.1 25 amb 6 d precipitate 40 20 mlwith agitation 0.1M NaAc, NaH2PO4 4.2M precipitate 4.1 3.8 5 amb 4 d 41a100 ml no agitation 0.1M NaAc, NaH2PO4 4.2M >95% 4.1 3.8 5 amb 7 d 41b 1ml, 50 ml or 10 l 0.1M NaAc, NaH2PO4 4.4M n.d. 4.1 n.d. 5 amb

E. Methods for Crystal Processing and Analysis Example 42 Washing ofCrystals

After formation of the crystals, a washing step without redissolving thecrystals is favourable. Therefore, after the crystallization process wasfinished, the crystal slurry was transferred into a centrifugation tubeand centrifuged at 500 to 1000×g for twenty minutes. The centrifugationwas performed at 4° C., but might also be performed at other feasibletemperatures, e.g. room temperature. After centrifugation, thesupernatant was discarded, and the crystal pellet was resuspended in abuffer containing around 4.2 M sodium dihydrogen phosphate in around 0.1M sodium acetate at a pH around 4.1. No measurable solubility of D2E7crystals in the washing buffer occurred, as analyzed by OD280. Thecentrifugation/resuspension steps were subsequently repeated for one tothree times, and after this washing procedure, the pellet wasresuspended and stored in a buffer containing around 4.2 M sodiumdihydrogen phosphate in around 0.1 M sodium acetate at a pH around 4.1.

Example 43 Analysis of Crystals by SDS-PAGE

To prove the protein character of the crystals, the crystals were washedwith a washing buffer as described in example 42. After assuring byOD280 that no more dissolved protein was present in the supernatantafter centrifugation, the supernatant was discarded, and the crystalswere subsequently dissolved in distilled water. OD280 measurement ofthis solution revealed that the crystals essentially consisted ofprotein, as the absorbance of the sample was now significantly higher asin the residual washing buffer. SDS-PAGE analysis of this solution ofredissolved crystals, when compared to an original D2E7 sample, showedthe same pattern.

F. Miscellaneous Examples

Concentration values given in the following examples are initial valuesreferring to the antibody solution and the crystallization solutionbefore mixing of the two solutions.

All pH values, if not described otherwise, refer to the pH of an acetatebuffer stock before it was combined with other substances, like thecrystallization agent.

All buffer molarities, if not described otherwise, refer to sodiumacetate concentrations in a stock solution before pH adjustment,typically performed using acetic acid glacial.

Example 44 Solid Crystallization Agent

D2E7 was used without exchanging the buffer (see Example 35).

D2E7 was brought to a concentration of around 10 mg/mL by diluting theliquid with Milli Q water.

Batch crystallization was performed by admixing around 500 μL of theprotein solution with an equal amount of acetate buffer (0.1 M, pH 4.1or 4.6, respectively) in a well of a 24 well plate. Subsequently, solidsodium dihydrogen phosphate dihydrate was added at six different ratiosto each pH setting: around 0.23 g, 0.27 g, 0.30 g, 0.33 g, 0.36 g and0.39 g. Thus, after complete dissolution of the crystallization agent,the concentration was around 1.5M to 2.5M in 0.2M steps. The wells weresubsequently sealed and the plate was agitated on a laboratory shakeruntil complete dissolution of the crystallization agent. Thereafter, the24 well plate was stored at ambient temperature without agitation.Microscopy of the plate was performed after five days.

Results:

Needle cluster-like crystals were found in the following seven batches:

-   -   acetate buffer pH 4.1 and sodium dihydrogen phosphate molarity        of around 2.1 M, 2.3M and 2.5M, respectively.    -   acetate buffer pH 4.6 and sodium dihydrogen phosphate molarity        of around 1.9M, 2.1 M, 2.3M and 2.5M, respectively.

Example 45 Different Buffer Preparation Protocol and Preparation ofCrystals

In this example, the acetate buffers were prepared as described in thefollowing: 3 g of acetic acid glacial were diluted with around 42 mL ofpurified water. The pH was adjusted with sodium hydroxide solution andthe volume adjusted to 50 mL. In this case, the total acetate amount isfixed at 1M (100 mM in the crystallization solution, or 50 mM in thecrystallization mixture) and not expanded by pH adjustment.

D2E7 was used without exchanging the buffer (see Example 35).

D2E7 was brought to a concentration of around 10 mg/mL by diluting theliquid with Milli Q water.

Batch crystallization was performed by admixing around 500 μL of theprotein solution with an equal amount of crystallization solution inwell of a 24 well plate. 500 μL of a particular crystallization solutionwas prepared by admixing acetate buffer, sodium dihydrogen phosphatestock solution, potassium dihydrogen phosphate stock solution and MilliQ water in a well. In this example, the acetate buffer molarity was 0.1M, and the acetate buffer pH was around 4.1 and 4.6, respectively.Sodium dihydrogen phosphate molarity was varied from around 3.4 M toaround 4.4 M in 0.2 M steps. The wells were subsequently sealed afterpreparation of the crystallization mixture to prevent water evaporation.Microscopy of the plate was performed after five days.

Results:

Needle cluster-like crystals were found in the following nine batches:

-   -   acetate buffer pH 4.1 and sodium dihydrogen phosphate molarity        of around 3.6 to 4.4, in 0.2 steps.    -   acetate buffer pH 4.6 and sodium dihydrogen phosphate molarity        of around 3.6 to 4.2, in 0.2 steps.

Example 46 Preparation of Encapsulated Crystals

Crystals as obtained in example 41 are positively charged as determinedvia zeta potential measurement using a Malvern Instruments Zetasizernano.

The crystals are washed and suspended in a buffer containing excipientswhich conserve crystallinity, and which has a pH that keeps the crystalscharged. Subsequently, an appropriate encapsulating agent is added tothe crystal suspension. In this context, an appropriate encapsulatingagent is a (polymeric) substance with low toxicity, biodegradability andcounter ionic character. Due to this counter ionic character, thesubstance is attracted to the crystals and allows coating. By thistechnique, the dissolution of crystals in media which do not contain anyother excipient maintaining crystallinity is preferably sustained.

Example 47 Preparation of Encapsulated/Embedded Crystals

Crystals are obtained as described in example 41.

The crystals are washed and suspended in a buffer containing excipientswhich conserve crystallinity.

The crystals can then be

-   -   embedded by drying the crystals and combining these dried        crystals with a carrier, e.g. by compression, melt dispersion,        etc.    -   encapsulated/embedded by combining a crystal suspension with a        carrier solution which is not miscible with water. The carrier        precipitates after removal of the solvent of the carrier.        Subsequently, the material is dried.    -   encapsulated/embedded by combining a crystal suspension with a        water miscible carrier solution. The carrier precipitates as its        solubility limit is exceeded in the mixture.    -   embedded by combining dried crystals or a crystal suspension        with a water miscible carrier solution.    -   embedded by combining dried crystals with a carrier solution        which is not water miscible.

G. Crystal Characterization

In the following section experiments that were performed to determinewhether crystalline monoclonal antibody D2E7 retains the bioactivitycharacteristic of never-crystallized D2E7 upon redissolution of thecrystalline material are summarized.

G1. Bioactivity Test with Murine L-929 Cells

a) General Method

The neutralizing effect of D2E7 solution against the cytotoxic effect ofrecombinant human TNF (rHuTNF) was determined. This involved incubatingmouse L-929 cells as indicator in a 96-well microtiter plate in thepresence of various D2E7 concentrations for 48 hours with a definedamount of rHuTNF at 37° C. The surviving cells were stained with crystalviolet. The intensity of color was measured by spectrophotometry in theindividual wells of the microtiter plate and evaluated. The IC₅₀ wasmeasured, i.e. the concentration of D2E7 which reduced the cytotoxiceffect of rHuTNF on L-929 cells such that 50% of the cells survived.

In a separate dilution box, starting from the 1 μg protein/mL dilutions,the 9 titer curve measuring points (curve dilutions) were preparedindividually in the dilution tubes for sample and reference standard.

The L-929 cell suspension to be used was diluted with medium to providea concentration of 60,000 cells/mL. Subsequently 100 μL per well of therespective cell concentration were pipetted into columns 1-11 of thetest plate. The wells in column 12 contained only 100 μL of medium each.Incubation was applied at 37° C. and 5% (v/v) CO₂ for 24 hours in thetest plate.

After 24 hours' incubation, 50 μL of each of the 9 titer curve dilutionswere transferred from the dilution box to the test plate for thereference standard or sample, i.e. for the reference standard to wellsin rows A-D in columns 1-9 and for the sample to the wells in rows E-Hin columns 1 to 9.

50 μL of medium were pipetted into column 10; and 100 μL each werepipetted into columns 11 and 12.

50 μL of TNF reference standard (12.5 ng protein/mL medium) werepipetted into the wells in column 1 to 10, row A to H, whereby column 10corresponded to the 100% lysis value (TNF control).

Column 11 was a 100% growth control, and column 12 contained no cellmaterial and thus acted as a blank. The final volume per well was 200μL.

Incubation of the test plates was performed for 48 hours at 37° C. andwith 5% CO₂. Following incubation for 2 days, the liquids from the testplate wells were discarded by turning quickly and giving a single,vigorous downward shake. Then 50 μL of crystal violet solution (0.75%crystal violet, 0.35% sodium chloride, 32.4% ethanol and 8.6%formaldehyde) were pipetted into each well. The solution was left in thewells for 15 minutes and then discarded as described above. Then theplates were washed and dried at room temperature for about 30 minutes.Subsequently, 100 μL of reagent solution (50% ethanol and 0.1% aceticacid) were pipetted into each well. Agitation of the plates (at about300 rpm for 15 min) produced an evenly colored solution in each of thewells.

The absorbance of the dye in the test plate wells was measured in aplate photometer at 620 nm. Individual values were plotted on a graph,with the absorbance (y axis) being plotted against the respectivedilution or concentration ng/mL (x axis) of antibody. From the4-parameter plot, the concentration was read off at which half the cellssurvive and half die (IC₅₀ value). This concentration was calculated byparameter 3 of the 4-parameter function of the curve data. The meanvalues of the reference standard concentrations were calculated. Therelative biological activity of the sample was calculated by dividingthe mean IC₅₀ value of the reference standard by the individual IC₅₀values of the sample and multiplication by 100%. The relative activitieswere then averaged.

b) Relative Activity for D2E7 Crystals

The test was performed as a comparison of the biological activity of thesample to that of a reference standard. The absorption values, plottedversus the concentration of D2E7 and assessed by a 4-parameter nonlinearregression, revealed the IC₅₀ values for the inhibition of the TNFeffect by the antibody. Since both samples were run in four repeats onone microplate this results in four IC₅₀ values for D2E7 referencestandard and sample respectively. Subsequently, the mean of the IC₅₀values of the reference standard was calculated and the relativeactivity of each repeat of the sample was assessed by dividing the meanIC₅₀ value of the reference standard by the relevant IC₅₀ value of thesample and multiplication by 100%.

The test of the sample (crystal suspension 2.7 mg/mL, prepared asdescribed in Example 36) revealed a relative biological activity of111%.

Thus, the sample can be considered as fully biologically active.

G2. Microscopic Characterization

In the following, data on microscopic characterization of crystals ofD2E7 will be presented.

a) Optical Analysis of mAb Crystal Batch Samples

After homogenization, aliquots of 1 to 10 μL sample volume were pipettedonto an object holder plate and were covered with a glass cover slide.The crystal preparations were assessed using a Zeiss Axiovert 25inverted light microscope equipped with E-PI 10× oculars and 10×, 20×and 40× objectives, respectively. Pictures were taken using a digitalcamera (Sony Cybershot DSC S75).

b) Optical Analysis of Vapor Diffusion Experiments, Assessment ofApproximate Crystal Sizes and Detection of Birefringence

For this purpose, a Nikon Labophot microscope was used, equipped withCFW 10× oculars and 4×, 10×, 20× and 40× objectives, respectively.

For assessment of vapor diffusion experiments, the sample drops in the24 well plates were screened.

Crystal sizes were assessed by transferring the microscopic picture ontoa computer screen by means of a JVC TK C1380 color video camera, and bymeasuring the length or diameter of representative needle-like or needlecluster-like crystals, applying the JVC Digital Screen Measurement Cometsoftware version 3.52a. Furthermore, the microscope was equipped with afilter set (polarizer and analyzer) to assess the birefringent behaviourof samples.

If the polarization directions of the polarizer and analyzer filters areset at a 90° angle relative to each other (“crossed polarizers”), nolight will pass through to the microscope eyepiece; the image willappear dark or black. If now a sample, which is placed into the lightbeam between the crossed polarizers, is capable of rotating thepolarization plane of the light, a distinct glimmering of the sampleagainst a dark background will be observed. This behaviour, termed“birefringence”, distinguishes ordered crystalline (anisotropic) fromunordered amorphous (isotropic) matter. As birefringence ischaracteristic for anisotropic matter, this glimmering appearance provesthe existence of crystalline matter. However, the absence ofbirefringence does not exclude the existence of crystalline matter, asthe crystals might also exhibit cubic symmetry and therefore beisotropic, like amorphous matter.

c) Results

In the attached FIGS. 1 to 4 representative pictures of D2E7 crystalsare presented.

FIG. 1 shows D2E7 crystals obtained by small-scale batch crystallizationaccording to Example 37 after 6 days at room temperature (5 mg/ml finalprotein concentration; Crystallization buffer: 4.2 M sodium dihydrogenphosphate in 0.1 M sodium acetate, pH 4.1). The crystals exhibitedbirefringence.

FIGS. 2 to 4 show D2E7 crystals obtained by large-scale batchcrystallization according to Example 41b.

Syringeability: A D2E7 crystal suspension 200 mg/mL protein incorporatedin crystals and formulated in a buffer containing 20% (m/v) PEG 4,000 issyringeable through a 27½ G needle.

G3. Birefringence

In order to demonstrate that Adalimumab crystallization in fact providescrystalline material its birefringence was analyzed.

Protein:

Diluted 70 mg/ml Adalimumab in formulation buffer with double-distilledwater to 10 mg/ml.

Precipitant:

4 M NaH₂PO₄ (dissolved powder in double-distilled water)

Method:

Micro batch crystallization in a hanging-drop tray with 2 ml compartmentwell, Mixed 500 μl protein solution with 500 μl protein; no NaOAc in thesolution.

Temperature:

24° C.

Technical Equipment for Birefringence Measurement:

Nikon SMZ1500 stereo dissecting microscope equipped with a Nikon CoolPixCCD camera. Crystal birefringence was photographed under crossedpolarizers. Magnification is approximately 200×.

Corresponding micrographs are shown in FIG. 5 A. A marked birefringenceof the clusters of Adalimumab needle-like crystals is observed. Thecolour of the crystals changes from blue to red, then back to blue, asthe orientation of the crystal needle axis rotates relative to the lightpolarization direction.

A further set of micrographs is depicted in FIGS. 5 B, C and D.

All images were taken with a Nikon Eclipse E600 POL microscope and aNikon DXM digital camera. Magnification is approximately 40×.

The image of FIG. 5B (grey) is taken with plane polars and shows theparticle morphology. The white crystals on the black background (FIG.5D) show birefringence and were taken with crossed polars. The blue andorange crystals on the purple background (FIG. 5C) show birefringenceand were taken with crossed polar and a red compensator or quarter waveplate.

H. Crystal Syringeability

In the following section, experiments were performed to determine thesyringeability of crystalline suspensions (in PEG) of monoclonalantibody D2E7 (10-200 mg/ml) using different gauge needles.

PEG Buffer: 20% PEG 4,000 m/v

12 mg/mL mannitol0.1 mg/mL polysorbate 801.305 mg/mL citric acid monohydrate0.305 mg/mL sodium citrate1.53 mg/mL di sodium hydrogen phosphate dehydrate0.86 mg/mL sodium di hydrogen phosphate dehydratepH was adjusted to 5.2 with sodium hydroxide

Syringe depletion (1 mL filling volume) was performed as it would bemanually by a patient in the course of administration. 20-27.5 G needlesizes were evaluated.

Syringes: 20/23/26 G:

Henke Sass Wolf GmbH 1 mL Norm-Ject syringes, equipped with

-   -   Henke Sass Wolf GmbH Fine-Ject® 20 G needles    -   Terumo® 23 G needles    -   Neopoint® 26 G needles

27.5 G:

BD HyPak SCF™ 1 mL long syringes, equipped with 27.5 G RNS needles 38800Le Pont du Claix

The results (FIG. 6) suggest that higher gauge needles provide a slowerdelivery of the crystals at at high concentrations.

I. Stability Data (SE HPLC, FT-IR)

In the following section, experiments were performed to determine thestability of crystalline suspensions of monoclonal antibody D2E7 (50 and200 mg/ml) over 12 month storage at 2-8° C.

Crystals suspended in medium as used in syringeability studies:

20% PEG 4,000 m/v

12 mg/mL mannitol0.1 mg/mL polysorbate 801.305 mg/mL citric acid monohydrate0.305 mg/mL sodium citrate1.53 mg/mL di sodium hydrogen phosphate dehydrate0.86 mg/mL sodium di hydrogen phosphate dehydratepH was adjusted to 5.2 with sodium hydroxide

SE-HPLC

50 mg/mL Adalimumab Crystal Suspension, Stable at 2-8° C. Over Time

Time point Aggregates (%) Monomer (%) Fragments (%) T0 1.3 98.5 0.2 1month 1.4 98.3 0.3 3 month 2.2 97.5 0.3 6 month 3.2 96.3 0.5 9 month 4.095.5 0.5 12 month  4.2 95.1 0.7200 mg/mL Adalimumab

Time point Aggregates (%) Monomer (%) Fragments (%) T0 1.3 98.5 0.2 1month 1.3 98.4 0.3 3 month 1.9 97.8 0.3 6 month 2.4 97.2 0.4 9 month 2.597.0 0.5 12 month  2.6 96.8 0.6

A Dionex HPLC system (P680 pump, ASI 100 autosampler, UVD170U) was usedfor stability analysis by SEC. D2E7 samples were separated on a GESuperose® 6 column, applying a flow rate of 0.5 mL/min. UV quantitation(detection) was performed at a wavelength of 214 nm. The running bufferconsisted of 0.15M sodium chloride in 0.02M sodium phosphate buffer pH7.5. IR spectra were recorded with a Confocheck system on a BrukerOptics Tensor 27. Liquid samples were analyzed using a MicroBiolyticsAquaSpec cell. Each sample was assessed performing at least twomeasurements of 120 to 500 scans at 25° C. Blank buffer spectra weresubtracted from the protein spectra, respectively. Protein secondderivative spectra were generated by Fourier transformation and vectornormalized from 1580-1720 cm⁻¹ for relative comparison. Redissolution ofcrystals was performed as follows: Crystal suspensions were diluted withHumira® commercial buffer to 10 mg/mL protein concentration. Bydecreasing PEG concentration crystals redissolved.

Blue—standard, Humira® after freeze/thawRed—redissolved crystals after 6 month storage at 25° C., 50 mg/mLGreen—redissolved crystals after 6 month storage at 25° C., 200 mg/mL

Results: FIG. 7 illustrates that there were no conformationaldifferences over 6 months of storage at 25° C.

J. Morphology

After 12 months of storage at 2-8° C. no significant morphologicalchanges were observed by light microscopy analysis of the crystals.

Aliquots of 1 to 10 μL sample volume were pipetted onto an object holderplate, diluted with formulation buffer (20% PEG) and covered with aglass cover slide. The preparations were assessed using a Zeiss Axiovert25 inverted light microscope equipped with E-PI 10× oculars and 10×, 20×and 40× objectives, respectively.

REFERENCES

-   Baldock Peter; Mills, Vaughan; Stewart, Patrick Shaw, Journal of    Crystal Growth (1996), 168(1-4, Crystallization of Biological    Macromolecules), 170-174.-   Connell, G. E., M. H. Freedman, et al. (1973). “Human IgG myeloma    protein crystallizing with rhombohedral symmetry.” Canadian Journal    of Biochemistry 51(8): 1137-41.-   Harris, L. J., S. B. Larson, et al. (1992). “The three-dimensional    structure of an intact monoclonal antibody for canine lymphoma.”    Nature (London, United Kingdom) 360(6402): 369-72.-   Huber, R., J. Deisenhofer, et al. (1976). “Crystallographic    structure studies of an IgG molecule and an Fc fragment.” Nature    264(5585): 415-20.-   Jen, A., Merkle, H. P. (2001), Diamonds in the rough: Protein    Crystals from a from a formulation perspective, Pharm. Res. (2001),    18, 11, 1483-   Jentoft, J. E., D. G. Dearborn, et al. (1982). “Characterization of    a human cryoglobulin complex: a crystalline adduct of a monoclonal    IgG and albumin.” Biochemistry 21(2): 289-294.-   Jones, H. B. (1848). “On a new substance occurring in the urine of a    patient with mollities ossium.” Philosophical Transactions of the    Royal Society. London 138: 55-62.-   McPherson, A. (1999). Crystallization of Biological Macromolecules.    Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press.-   Mills, L. E., L. R. Brettman, et al. (1983).    “Crystallocryoglobulinemia resulting from human monoclonal    antibodies to albumin.” Annals of internal medicine 99(5): 601-4.-   Nisonoff, A., S. Zappacosta, et al. (1968). “Properties of    crystallized rabbit anti-p-azobenzoate antibody.” Cold Spring Harbor    Symposia on Quantitative Biology 32: 89-93.-   Putnam, F. W. (1955). “Abnormal human serum globulins.” Science    (Washinaton, D.C. United States) 122: 275-7.-   Rajan, S. S., K. R. Ely, et al. (1983). “Three-dimensional structure    of the Mcg IgG1 immunoglobulin.” Molecular Immunology 20(7): 787-99.-   Sarma, V. R., E. W. Silverton, et al. (1971). “Three-dimensional    structure at 6 Ang. Resolution of a human gG1 immunoglobulin    molecule.” Journal of Biological Chemistry 246(11): 3753-9.-   Shenoy, B., C. P. Govardhan, et al. (2002). Pharmaceutical    compositions comprising crystals of polymeric carrier-stabilized    antibodies and fragments for therapeutic uses. PCT Int. Appl. WO,    (Altus Biologics Inc., USA). 173 pp.-   Terry, W. D., B. W. Matthews, et al. (1968). “Crystallographic    studies of a human immunoglobulin.” Nature 220(164): 239-41.-   von Bonsdorf, B., H. Groth, et al. (1938). “On the Presence of a    High-molecular Crystallizable Protein in Blood Serum in Myeloma.”    Folia Haematologia 59: 184-208.-   Yang, M. X., B. Shenoy, et al. (2003). “Crystalline monoclonal    antibodies for subcutaneous delivery.” Proceedings of the National    Academy of Sciences of the United States of America 100(12):    6934-6939.

1. A batch crystallization method for crystallizing an anti-hTNFalphaantibody, comprising the steps of: (a) providing an aqueous solution ofsaid antibody in admixture with an inorganic phosphate salt ascrystallization agent; and (b) incubating said aqueous crystallizationmixture until crystals of said antibody are formed.
 2. Thecrystallization method according to claim 1, wherein said aqueouscrystallization mixture a) has a pH in the range of about pH 3 to 5; orb) comprises a buffer.
 3. (canceled)
 4. The crystallization methodaccording to claim 2, wherein said buffer comprises an acetate buffer.5-8. (canceled)
 9. The crystallization method according to claim 1,wherein the phosphate salt is selected from the group consisting of a) ahydrogenphosphate salt; b) an alkali metal salt; and c) a mixture of atleast two different alkali metal salts. 10-12. (canceled)
 13. Thecrystallization method according to claim 1, wherein at least one of thefollowing additional crystallization conditions are met: a) incubationis performed for between about 1 hour to about 60 days; b) incubation isperformed at a temperature between about 4° C. and about 37° C.; c) theantibody concentration is in the range of about 0.5 to about 100 mg/ml.14. The crystallization method according to claim 13, further comprisingthe step of drying said crystals.
 15. (canceled)
 16. The crystallizationmethod according to claim 13, wherein the batch volume is in the rangeof about 1 ml to 20.000 liters.
 17. A crystal of an anti-hTNFalphaantibody, obtainable by a crystallization method according to claim 1.18. A crystal of an anti-hTNFalpha antibody, with the proviso that saidantibody is not INFLIXIMAB.
 19. The crystal of claim 17 having aneedle-like morphology with a maximum length l of about 2-500 μm and anl/d ratio of about 3 to
 30. 20. The crystal of claim 18 having aneedle-like morphology with a maximum length l of about 2-500 μm and anl/d ratio of about 3 to
 30. 21. The crystal according to claim 17,wherein said antibody is selected from a group consisting of a) apolyclonal antibody; b) a monoclonal antibody; c) a human antibody; d) ahumanized antibody; e) a non-glycosylated antibody; and f) a chimericantibody. 22-25. (canceled)
 26. The crystal according to claim 18,wherein the antibody is an isolated human antibody; selected from thegroup consisting of a) an isolated human antibody that dissociates fromhTNFalpha with a Kd of 1×10⁻⁸ M or less and a Ka rate constant of 1×10⁻³s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes hTNFalpha cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less; b) an isolated human antibody with thefollowing characteristics: i) dissociates from human TNFalpha with aKoff rate constant of 1×10-3 s-1 or less, as determined by surfaceplasmon resonance; ii) has a light chain CDR3 domain comprising theamino acid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by asingle alanine substitution at position 1, 4, 5, 7 or 8 or by one tofive conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8and/or 9: iii) has a heavy chain CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by oneto five conservative amino acid substitutions at positions 2, 3, 4, 5,6, 8, 9, 10, 11 and/or 12; c) an isolated human antibody with a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 and a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 2; and d) the adalimumab. 27-29. (canceled)30. A pharmaceutical composition comprising: (a) crystals of ananti-hTNFalpha antibody according to claim 17, and (b) at least onepharmaceutical excipient; which composition is provided in as solid,semisolid or liquid formulation, each formulation containing saidantibody in crystalline form. 31-35. (canceled)
 36. An injectable liquidcomposition comprising anti-hTNFalpha antibody crystals according toclaim 17 and having an antibody concentration in the range of about 10to 400 mg/ml.
 37. A crystal slurry comprising anti-hTNFalpha antibodycrystals according to claim 17, having a antibody concentration greaterthan about 100 mg/ml.
 38. A method for treating a mammal comprising thestep of administering to the mammal an effective amount of antibodyanti-hTNFalpha crystals according to claim
 17. 39. A method for treatinga mammal comprising the step of administering to the mammal an effectiveamount of the composition according to claim
 30. 40. (canceled)
 41. Amethod of treating a TNFalpha-related disorder in a subject, whichmethod comprises administering a therapeutically effective amount ofantibody crystals according to claim
 17. 42-44. (canceled)